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Intramolecular heterogeneity of IgM antibodies

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Title:
Intramolecular heterogeneity of IgM antibodies
Creator:
Giles, Robert Clay, 1952-
Publication Date:
Language:
English
Physical Description:
x, 96 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Antibodies ( jstor )
Binding sites ( jstor )
Cell lines ( jstor )
Gels ( jstor )
Haptens ( jstor )
Hybridomas ( jstor )
Kidney dialysis ( jstor )
Ligands ( jstor )
Molecular chains ( jstor )
Molecules ( jstor )
Dissertations, Academic -- Immunology and Medical Microbiology -- UF ( mesh )
Hybridomas ( mesh )
Immunoglobulin M ( mesh )
Immunoglobulins, Heavy-Chain ( mesh )
Immunology and Medical Microbiology thesis Ph.D ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida.
Bibliography:
Bibliography: leaves 90-95.
General Note:
Photocopy of typescript.
General Note:
Vita.
Statement of Responsibility:
by Robert Clay Giles.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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Full Text













INTRAMOLECULAR HETEROGENEITY OF IgM ANTIBODIES


BY

ROBERT CLAY GILES








A DISSERTATION PRESENTED TO THE GRADUATE
COUNCIL OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY



UNIVERSITY OF FLORIDA


1982




INTRAMOLECULAR HETEROGENEITY OF IgM ANTIBODIES
BY
ROBERT CLAY GILES
A DISSERTATION PRESENTED TO THE GRADUATE
COUNCIL OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1 982


"Take instruction, and not silver,
And knowledge rather than choicest gold.
For wisdom is better than jewels;
And all desirable things can not compare
with her."
Proverbs 8:10-11 (NAS)
The Bible


ACKNOWLEDGEMENTS
First, thanks to Dr. Bruce Glick for his encouragement
to me that I pursue this undertaking and for his assistance
in allowing it to become possible. I would like to acknow
ledge the continued support and encouragement of my'vi sor
Dr. William Clem. His experience and perspective in solving
scientific problems have been a major contribution to my
learning process. I am appreciative of. both his tutorage
and his friendship. I would also like to extend my appre
ciation to Dr. Kenneth Berns as Chairman of the Department
of Immunology and Medical Microbiology and the other mem
bers of my committee, Dr. George Gifford, Dr. Bill Holloman,
and Dr. Paul Klein, for their time and advice. I would also
like to thank Dr. David Klapper for serving as an advisor
for this study and for his collaboration.
Thanks go out to my fellow graduate students, Terry
Van Dyke, Jim Rusche, and Tom Rowe, for helping to make my
short stay in Gainesville a memorable one. Special thanks
to Erv Faulmann and Marie Hoo'ver for being my friends
through it all.
Thanks to my father and my mother for always giving
of themselves unselfishly and for their continued support
and encouragement during this endeavor of my life. I
would like to thank my wife and closest friend, Jansen,
i i i


for her love and sacrifice during this time of our lives.
Without her understanding and support this work would not
have been possible. I would like to thank my son Clay
for the added joy he has brought to our lives and for
*
not keeping us up too many nights. I look forward to
growing and learning together as a family.


TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS . iii
LIST OF TABLES vi
LIST OF FIGURES vii
ABSTRACT ix
CHAPTER I BACKGROUND 1
CHAPTER II INTRASUBUNIT HOMOGENEITY IN HETERO
GENEOUS IgM ANTIBODIES TO THE DNP
MOIETY DERIVED FROM A MURINE HYBRIDOMA
CELL LINE
Introduction 11
Materials and Methods 12
Results 14
Discussion 34
CHAPTER III INTRAMOLECULAR HETEROGENEITY OF TWO
IgM ANTIBODIES TO THE DNP MOIETY
DERIVED FROM MURINE HYBRIDOMA CELL
LINES
Introduction 39
Materials and Methods 41
Results 49
Discussion 78
CHAPTER IV SUMMARY AND CONCLUDING REMARKS 86
REFERENCES 90
BIOGRAPHICAL SKETCH 96
v


LIST OF TABLES
TABLE PAGE
1. Amino Terminal Sequences of Light Chains
from Unfractionated 19S and Active and
Inactive 7S Reductive Subunits from 14PAF . 26
2. Amino Terminal Sequences of Heavy and Light
Chains of IgM Antibody to DNP from a Hybridoma
Line NP3-17 Cl -20 55
3. Comparison of Hapten Elution Profiles of Mildly
Reduced SP2/0 1-64 C1 -12 IgM Antibody from Tm-
and Non-Tm-treated Culture Supernatants ... 75
4. Immunoprecipitation of Tunicamycin (Tm)-
treated and Non-Tm-treated Culture Supernatants
and Cel 1 Lysates 76


LIST OF FIGURES
FIGURE PAGE
1. Equilibrium dialysis of mouse hybridoma
protein 14PAF with DNP-e-aminocaproate .... 17
2. Equilibrium dialysis of DNP-e-aminocaproate
of 7S subunits from 14PAF 20
3. Step-wise elutions of protein 14PAF from
DNP-lys-sepharose affinity column using
four concentrati00^.of hapten 22
4. Alkaline urea polyacrylamide gel electro
phoresis of isolated light chains from
unfractionated (A), inactive (B), and active
(C)7Ssubunits 25
5. Equilibrium dialysis with DNP-e-aminocaproate
of 14PAF recombinants using either heavy chains
from active subunits (AH) or inactive subunits
(IH) and light chains from active subunits
(AL) 30
6. SDS-PAGE of recombinants formed using heavy
chains from either active or inactive subunits
combined with an equimolar amount of light
chains consisting of 60% from active subunits
and 40% from inactive subunits 33
7. Mild reduction profile of 19S IgM from
hybridoma SP2/0 1-64 C1 -12 using increasing
amounts of 2-ME from left to right 44
8. Equilibrium dialysis of 19S IgM from
hybridoma SP2/0 1-64 C1 -12 with DNP-e-amino
caproate 52
9. Alkaline urea polyacrylamide gel electro
phoresis of isolated light chains from (A)
SP2/0 1-64 Cl-12 and (B) NP3-17 Cl-20 .... 54
10. Equilibrium dialysis using DNP-e-aminocaproate
of NP3-17 Cl-20 "7S" fractions resulting from
mild reduction at dilute protein concentra
tions (< 1 mg/ml ) 60
11. 10% polyacrylamide gel electrophoresis (SDS)
of NP3-17 Cl-20 "7S11 62
v i i


FIGURE
PAGE
12. 10% polyacrylamide gel electrophoresis of
timed trypsin digestions of SP2/0 1-64 Cl-12
7S reductive subunits 64
13. Sephadex G-200 elution profile of SP2/0 1-64
Cl-12 Fab's obtained after trypsinization
for five hours 67
14. 10% polyacrylamide gel electrophoresis (SDS)
of unfractionated (A) and active (C) Fab's
obtained from five hour trypsinization of
SP2/0 1-64 Cl-12; (B) contains inactive, non-
Fab material 69
15.Equilibrium dialysis of Fab fragments
obtained from a five hour trypsinization
of SP2/0 1-64 Cl-12 with DNP-e-aminocaproate .
1 6
Autoradiogram of specifically purified
^^S-methi on i ne molecules from supernatants
or cell lysates of Tm- or non-Tm-treated
SP2/0 1-64 Cl-12 cells
71
74
v i i i


Abstract of Dissertation Presented to the Graduate
Council of the University of Florida in Partial
Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
INTRAMOLECULARi HETEROGENEITY OF IgM ANTIBODIES
BY
Robert Clay Giles
May, 1982
Chairman: Dr. L. William Clem
Major Department: Immunology and Medical Microbiology
A hybridoma line (14PAF) secreting 19S IgM antibodies
reactive with the DNP moiety was derived from a fusion of
DNP-primed murine spleen cells with the myeloma line P3-
X63-Ag-8. The failure to demonstrate ten binding sites per
pentameric molecule (six were measured) was attributable to
the presence of two different light (L) chains. Analysis
of the reductive subunits indicated the presence of two
types. One type (designated active) contained an average
of two homogeneous ligand binding sites per subunit and did
not contain L chains normally found in MOPC-21 the myeloma
protein secreted by the P3 myeloma line. The other type of
subunit (designated inactive) did not contain any DNP
binding sites and contained only MOPC-21 L chains. Recom
bination studies with y and L chains from each type of
reductive subunit indicated that the noncovalent assembly
of the 2 p 2 L chain subunits was restricted by the L chains


in such a way that the recombinant subunits were homogen
eous in terms of L chains. Equilibrium dialysis studies
with active homogeneous recombinant subunits indicated the
presence of but one ligand binding site per 2 p-2 L chain
subunit.
Two additional murine anti-DNP IgM hybridoma anti
bodies were derived using the non-immunoglobulin producing
myeloma lines P3-X63-Ag8.653 and SP2/0-Ag-l4. These IgM
antibodies, NP3-17 Cl-20 and SP2/0 1-64 Cl -12, displayed
an average of five high affinity binding sites for the DNP
moiety. Reductive subunits of each of these proteins
absorbed to and were hapten eluted from an affinity column.
These subunits, when examined by equilibrium dialysis, each
contained an average of one high affinity binding site.
Structural analysis of these molecules indicated each to be
homogeneous. Results obtained from trypsin hydrolysis of
each molecule as well as studies performed with reductive
halfmers (H-L) indicated that differences in conformation
of the binding sites may account for the observed binding
heterogeneity.
The results of the chain recombination studies with
14PAF closely mimic the natural situation that occurs with
NP3-17 Cl-20 and SP2/0 1-64 Cl-12. These findings strongly
implicate the mechanism of assembly as a possible factor in
the generation of conformational differences that could ac
count for the intramolecular heterogeneity of ligand binding
seen with certain IgM molecules.
i x


CHAPTER I
BACKGROUND
The structure and physiochemica1 properties of mammal
ian IgM immunoglobulins have been elaborated in considerable
detail largely through the study of homogeneous IgM myeloma
proteins. These studies demonstrate that secreted mammalian
IgM antibodies are pentameric molecules containing ten
heavy-light chain pairs and thus, by analogy with IgG
antibodies, should contain ten equivalent ligand binding
sites per molecule (reviewed 1). Although several studies
employing structurally homogeneous IgM monoclonal proteins
have demonstrated the presence of ten homogeneous, rela
tively low affinity binding sites per molecule (2,3),
numerous other studies with conventional IgM antibodies
have indicated considerable heterogeneity of ligand binding
with valences of less than ten. In several instances
valences of less than ten were attributable to steric
factors related to antigen size. For example, in one
study the measured valences of IgM antibodies to dextran
progressively decreased as the size of the poly-glucosan
ligand was increased (4). Similarly in another study a
human IgM myeloma protein which bound human IgG was observed
to have an effective valence of five per intact pentamer
(one per subunit) whereas each of the ten Fabp fragments
contained an active binding site (5). In other cases,
-1-


2
haptens which are not likely to impose steric limitations
on the antibody combining sites were employed. In this
latter context it is important to point out that an average
of five high and five low affinity binding sites .has fre
quently been observed in a variety of species (6,7,8,9).
One study utilizing an unusual IgM subunit containing only
a single heavy-light chain (10) demonstrated that 50% of
these subunits were retained by an immunoadsorbant column
previously used to isolate this heterogeneous antibody from
serum. Since it was also shown that greater than 90% of
the 7S subunits of this IgM antibody were retained by an
immunoadsorbant, the hypothesis was suggested that hetero
geneity must exist within individual 7S subunits. However
due to the fact that the IgM was not homogeneous, critics
explained the data by the presence of different populations
of high and low affinity IgM molecules', half of which lack
sufficient affinity as heavy-light chain pairs to be
retained by an immunoadsorbant.
In terms of resolving this issue there would seem to
be two different, but not necessarily exclusive, possibil
ities. The first is that ligand binding heterogeneity is
a consequence of differences between antibodies in a popu
lation, i.e., intermolecular differences due presumably to
differences in antibody primary structure. The second
possibility is that ligand binding heterogeneity reflects
differences within individual antibody molecules, i.e.,
intramolecular differences. While the first possibility


3
is likely the case in some circumstances wherein obviously
structurally heterogeneous antibodies are employed, the
frequent finding of an average of half high and half low
(% 11 as high as those with high affinity) affinity sites
suggests consideration of the second possibility. Further
more, one attempt at separating the two populations of
binding sites suggested that both were on the same IgM
molecule (11), hence adding credence to the possibility
of intramolecular heterogeneity.
The solution to this question has, to a large part, been
elusive due to difficulties in obtaining sufficient amounts
of structurally homogeneous IgM antibodies. A potential
approach to this question was offered by the observations
that the physicochemical properties of immunoglobulins from
lower vertebrates indicate that such animals may be
restricted to but one immunoglobulin isotype analogous to
IgM (reviewed, 12,13). Furthermore, studies using sharks
indicated that certain antigens, especially A-variant
streptococci, can elicit the production of very large
amounts of relatively homogeneous 19S antibodies (up to
10 mg/ml serum) (14,15). Although it has not been possible
to isolate a suitable hapten for studying affinities and
valences with this antigen (16), the finding in sharks of
reasonably good 19S antibody responses to the capsular
polysaccharide of pneumococcal cells and to the DNP hapten
covalently coupled to streptococcal cells provided sufficient
material for limited studies. The results of these studies
with shark IgM antibodies are summarized below.


4
Shark 19S antibodies to the Type III (S^) pneumococcal
capsular polysaccharide were isolated from immune sera by
affinity chromatography and subjected to equilibrium
dialysis using the hexasaccharide hapten. The results
demonstrated several important points: (1) The IgM anti
bodies contained an average of ten combining sites per
molecule, (2) in all cases, the antibodies showed marked
heterogeneity of affinities, (3) the antibodies were all
of low average affinities and (4) there was. no increase in
the average affinity of the antibodies isolated from any
single animal for periods up to twelve months after initial
immunization. In order to determine if a single IgM mole
cule contained ten equivalent combining si tes, the anti
bodies isolated from several animals were fractionated by
liquid isoelectric focusing. Equilibrium dialysis experi
ments using focused fractions showed the presence of ten
functionally identical combining sites per 19S molecule.
As a proof of the structural homogeneity of focused
fractions, antibodies were mildly reduced, separated into
H and L chains, recombined to 7S subunits (2H-2L chains),
and tested for combining sites by equilibrium dialysis.
The results indicated that these 7S recombinants of focused
antibody fractions each contained two binding sites iden
tical to those of the intact antibody whereas heterogeneous
(unfocused) recombinants or isolated H and L chains failed
to show any binding activity. The conclusion from this
study is that the heterogeneity of ligand binding exhibited


5
by nurse shark 19S antibodies to the capsular polysac
charide of the Type III pneumococcus can be attributed
to intermolecular heterogeneity most likely at the primary
structural level (17).
In contrast to the results obtained in sharks with the
pneumococcal antigen, those obtained with antibodies to the
DNP moiety dictate a quite different conclusion (18),
Equilibrium dialysis studies using the hapten DNP-e-amino-
caproate with affinity purified nurse shark 19S antibodies
to DNP demonstrated several important points: (1) The 19S
antibodies exhibited heterogeneity of ligand binding with
an average of five high and five low affinity sites per
molecule, (2) the affinities of the low affinity sites were
approximately 1% of those of the high affinity sites, and
(3) no evidence for increased affinities was seen for up to
twenty-one months of immunization. To study the basis for
the ligand binding heterogeneity of the shark 19S anti
bodies to DNP, several different approaches were employed.
The results of studies with subunits and proteolytic frag
ments showed that neither steric hindrance nor allosteric
effects could account for the observed heterogeneity, i.e.,
the two forms of binding sites were on separable Faby frag
ments. In fact these results strongly suggested that both
types of combining sites were present within individual
reductive subunits. Preparative liquid isoelectric focus
ing was used in an attempt to separate structurally homo
geneous 19S antibodies. The results from equilibrium


6
dialysis studies indicated that each of 16 different focused
preparations contained an average of five high and five low
affinity sites-. These results seemingly argue strongly
against the possibility that the observed heterogeneity was
due to intermolecular heterogeneity. It seems highly
improbable that each of sixteen different isoelectrically
focused anti-DNP preparations would fortuitously be composed
% *
of equimolar mixtures of 19S molecules, half of which have
ten equivalent ligand binding sites approximately 100 times
higher in affinity than those of the remaining low affinity
population. It would seem more likely that the isoelectric
focusing technique actually separated structurally homo
geneous antibodies. This interpretation .is supported by the
results of recombination studies. Heavy and light chains
from focused molecules yielded 7S recombinants with high and
low affinity sites, albeit in low yields, indistinguishable
from their putative counterparts on the intact molecule.
Since heterogeneous recombinants resulted in binding sites
of only low affinities, it seems likely the focused prepara
tions were structurally homogeneous and hence the observed
ligand binding heterogeneity must be an intramolecular
phenomenon (19).
The presence of binding sites of two different affin
ities on a single antibody molecule can be explained by
primary structural and/or conformational differences between
the two types of sites. Although the presence of amino acid
sequence heterogeneity in the heavy and/or light chains of a


7
single IgM molecule seems unlikely, the lack of amino acid
sequence data on the shark IgM antibodies makes it impossible
to a^ priori rule out this explanation, Despite this
unclarified point, evidence from another experimental
approach seems to favor the latter possible explanation
mentioned above. Shark 19S antibodies to the DNP moiety
(exhibiting five high and five low affinity sites) and to
the polysaccharide (exhibiting ten low affinity sites)
were each treated with 5 M guanidine-HCl and studied by
equilibrium dialysis with the appropriate hapten after
removal of the denaturing solvent. The results showed this
treatment had no effect on the number or affinity of sites
on the antibodies to the polysaccharide but had a consid
erable effect on the antibodies to DNP. In fact the data
obtained with these latter antibodies indicated linear
Scatchard plots that readily extrapolated to a valence of
ten low affinity sites. Thus it appears that the guanidine-
HC1 treatment converted the five high affinity sites to low
affinity ones. This was presumably accomplished by causing
conformational changes in or near the high affinity sites,
Unfortunately sufficient amounts of shark antibodies were
not available to perform detailed studies of the conforma
tional relationships between Faby fragments containing high
or low affinity sites.
In light of the suggestion that the intramolecular
heterogeneity of ligand binding by some shark (and perhaps
other species) IgM antibodies to the DNP moiety may result


8
from intramolecular conformational differences, it seems
appropriate to speculate on possible mechanisms responsible
for these putative differences. One possible explanation
is that IgM molecules may be assembled intracel1ularly by
two different mechanisms depending upon the cell involved.
Since the data discussed above suggest these differences
are in fact intrasubunit differences, it is conceptually
sound to' suggest that those lymphocytes secreting IgM
antibodies with ten homogeneous sites may do so by assembling
the subunits (2H-2L) from halfmers (H-L) as is the case for
a relatively limited number of IgM myeloma proteins studied
(20,21). In this case it would be predicted that the sub
units should exhibit symmetry of ligand binding. On the
other hand, those cells secreting IgM antibodies with half
high and half low affinity sites may do so by assembling
the subunits in a different manner, i.e., either H-H+H-H-L-*-
L-H-H-L or H-L+H-H-L+L-H-H-L. If these latter modes of
assembly exist, as is the case with certain other immuno
globulin iso types (22), it is conceivable (although admit
tedly antidogma) that the formation of the first H-L chain
pair may somehow influence the conformation of the second
pair so as to result in a different affinity site. A priori,
one cannot tell which (high or low affinity) site would be
formed first.
Since the suggestion of intramolecular conformational
differences may seem to some to reflect too much "antidogma,"
it would be appropriate here to briefly consider this issue.


9
There are several reports in the literature which document
the existence of small but detectable conformational dif
ferences between Fab fragments derived from specific anti
bodies and immunoglobulins (23,24). Admittedly these
proteins were heterogeneous (intermolecular) in primary
structure and hence the existence of such conformational
differences may not be surprising, Furthermore, while the
relatively recent surge of X-ray crystallographic data has
indicated considerable 3-dimensional similarities between
several homogeneous Fab fragments, there are also indica
tions of slight differences (reviewed 25), Perhaps the most
important observations that could be cited in support of the
possible existence of intramolecular conformational hetero
geneity are those of Edmundson and colleagues (26) with the
myeloma protein and dimer Bence-Jones protein of patient Meg.
These data indicate rather conclusively that different
conformations of a polypeptide chain (L chains in their
study) can be derived from the same amino acid sequence
depending upon the other chain to which the peptide is
paired. Similar precedents have been reported for the
crystalline structures of dimeric insulin (27) and chymo-
trypsin (28). Thus it does not seem absurd at this point to
suggest that such conformational differences could exist and
be functionally important within structurally homogeneous
IgM molecules. Finally, since part of our rationale for
suggesting the role of intramolecular conformational differ
ences to explain the apparent intramolecular heterogeneity


10
of ligand binding of some IgM antibodies was based upon the
results obtained with guanidine-HCl treated antibodies, the
results of Richards and coworkers with mouse IgA myeloma
protein 460 become pertinent (29), Their data indicated
that this protein, possessing two ligand binding specifi
cities, could be rendered unreactive with one ligand (DNP)
by the guanidine-HCl treatment whereas the affinity for the
other (menadione) was unaffected, Presumably this partial
loss in function resulted from an intracombining site
conformational change. It is unknown if such a change
could be detected by the methodology available.


CHAPTER II
INTRASUBUNIT HOMOGENEITY IN HETEROGENEOUS
IgM ANTIBODIES TO THE DNP MOIETY DERIVED
FROM A MURINE HYBRIDOMA CELL LINE
Introduction
In light of the immunologic importance of IgM it
becomes imperative to develop approaches for obtaining
sufficient amounts of structurally homogeneous mammalian
IgM antibodies with specificities for defined ligands in
order to clearly resolve the question of intramolecular
heterogeneity. The approach that seemed most promising and
was undertaken involved cell fusions (hybridomas). Chapter
II describes the initial results obtained with one murine
hybridoma line secreting 19S IgM antibodies reactive with
the DNP moiety. Although the secreted IgM product was a
structurally heterogeneous molecule (due to the presence of
two different light chains) it appeared to be assembled in
a random fashion from two pools of homogeneous subunits.
Furthermore i_n vi tro recombination studies with the
component polypeptide chains revealed the surprising
findings that a) the subunit homogeneity was L chain
directed and b) each recombinant subunit exhibited but
one active site.


Materials and Methods
Hybridomas
The fusion techniques described by Galfre' et al .
(30) and Gerhard et al. (31) were employed with some modi-
O
fications. Briefly 1 x 10 BALB/c spleen cells (mice
injected 3 days previously with 50 yg 2,4-dinitrophenyl-
ficoll ) and 1 x 10^ myeloma cells (P3-X63-Ag8; derived by
Cotton and Milstein) (32) were mixed and centrifuged at
300g for 8 minutes. Cells were resuspended for fusion in
1 ml of a 50% solution (v/v) of polyethylene glycol (PEG-
1000, J.T. Baker Chemical Co.) in Dulbecco's Minimal
Essential Medium (DMEM, Gibco). This suspension was
further diluted at a rate of 6 ml/min with DMEM over a
period of 5 minutes. The cells were washed and resuspended
in 35 ml of the selective medium which contains hypoxan-
thine, aminopterin, and thymidine (33). The cells were
distributed in 100 yl aliquots into 96 well microtiter
plates (Falcon Microtest II) and incubated at 37C in a
humidified 5% CO^-95% air mixture. After substantial
growth (^ 10-12 days) supernatants were screened for IgM
antibodies to the DNP moiety using a radioimmunoassay (34).
Cultures positive for anti-DNP antibody were cloned by
limiting dilution in soft agarose (35). Individual posi
tive clones ('v 5 x 10 cells) were injected into BALB/c
mice primed with pristane to obtain ascitic tumors. One
such clone, designated 14PAF, was selected for the studies
reported here.


13
Immunochemical Procedures
Mouse antibodies to the DNP moiety were purified from
ascitic fluid by affinity chromatography, freed of hapten,
3
and examined by equilibrium dialysis against H-DNP-e-
aminocaproate as described previously (18). For calculating
protein concentrations of the pentameric IgM antibodies, a
molecular weight of 900,000 daltons and an extinction
coefficient (Eoori ) of 11.9 were assumed.
In one experiment 14PAF was subjected to 5.0 M
guanidine-HCl for one hour at room temperature. The
guanidine-HCl was removed by dialysis against Tris buffered
saline (0.15 M NaCl 0.01 M Tris-HCl, pH 7.4). These
guanidine-HCl treated antibodies were subjected to equili
brium dialysis as previously described.
Mildly reduced 7S subunits of protein 14PAF were pre
pared by subjecting purified 19S material to reduction with
0.1 M 2-mercaptoethanol in 0.5 M Tris-HCl, pH 8.0, for one
hour at 22C followed by alkylation with 0.15 iodoacetamide
for one hour on ice. Gel filtration under nondenaturing
conditions (Sephadex G-200 equilibrated with 0.15 M NaCl,
0.01 M Tris-HCl, pH 7.4) indicated that > 95% of such re
duced and alkylated 7S material eluted in a volume expected
to contain ^180,000 dalton proteins; analysis under
denaturing conditions without additional reduction indi
cated that >90% of the 7S subunits dissociated into equi
molar H and L chains. A small percentage of halfmer (H-L)
molecules was also detected. Mildly reduced and alkylated


14
H and L chains and halfmers were obtained by gel filtering
the 7S subunits on Agarose A5M columns equilibrated with
5 M guanidine-HCl containing 0.01 M iodoacetamide.
Recombinant molecules were prepared from mildly reduced
and alkylated H and L chains as described previously (17).
Briefly, the desired amounts of separated H and L chains
were mixed in 5 M guanidine-HCl containing 0.01 M iodoace-
tamide and concentrated by positive pressure dialysis to
^ 5 mg/ml while dialysing against Tris buffer, pH 7.4.
These recombinants were gel filtered under nondenaturing
conditions and >90% of the UV absorbing material eluted in
a volume expected to contain 180,000 dalton proteins;
electrophoresis of these recombinants in SDS polyacryla
mide gels indicated the--presence of equimolar H and L
chains (see Figure 5 for example). Alkaline-urea gel
electrophoresis of extensively reduced L chains was per
formed by the method of Reisfield and Small (36). SDS-
polyacrylamide gel electrophoresis was performed according
to the method of Laemmli (37). Amino terminal sequence
studies were performed by Edman degradation using an auto
mated Beckman sequenator. PTH derivatives were identified
by high pressure liquid chromatography (38).
Results
The hybridoma line (14PAF) used here readily prolif
erated as ascitic tumors in pristane-primed BALB/c mice and
yields of 10-20 ml ascitic fluid were obtained from indivi
dual mice. Affinity chromatography of these ascitic fluids


on TNP-lys-sepharose yielded from 5-10 mg/ml antibody.
These isolated antibodies were considered to be exclusively
19S IgM(K) based upon sedimentation velocity measurements,
immunodiffusion analysis with commercial antisera and
SDS-gel electrophoresis studies (data not shown).
Equilibrium dialysis of the isolated 19S antibody
against the hapten 2,4-dinitrophenyl-e-aminocaproate
yielded the Scatchard plot depicted in Figure 1. These
data indicate the hybridoma derived IgM antibody contained
an average of about six binding sites per molecule with an
affinity of 2 x 10^M-^. Furthermore, Sips analysis indi
cated a heterogeneity index of 0.98 which presumably
reflected a high degree of homogeneity in the binding
constants of the sites being detected. Equilibrium dialysis
of the 19S antibody treated with 5.0 M Gn-HCl demonstrated
an identical Scatchard plot to the untreated antibody
(see Figure 1).
In order to determine if the observation of about six
binding sites per molecule was due to some peculiar steric
effects, reductive 7S subunits of protein 14PAF were prepared
and studied by equilibrium dialysis. As can be seen in
Figure 2, these 7S subunits appeared to exhibit a ligand
binding pattern identical to that observed with the parent
pentameric molecule, i.e., M 2 sites with an affinity of
% 2 x 10^M~^ per every two H-L chain pairs. These reductive
subunits were then subjected to affinity chromatography on
DNP-1ysine-sepharose. Approximately 55-60% of the material,


Figure 1 .
Equilibrium dialysis of mouse hybridoma protein
14 PAF with DNP-e-aminocaproate.


o


18
designated as active, absorbed and was hapten-eluted from
the affinity matrix; the other 40-45%, designated as
inactive, did not absorb. Each of these populations of
subunits was examined by equilibrium dialysis. As pre
sented in Figure 2, the active subun its .exhibited two homo
geneous binding sites identical with respect to affinity to
those seen in the unfractionated material. The inactive
subunits contained no demonstrable binding sites for DNP'.'
The finding that both the active and inactive subunits
could be derived from functional IgM molecules prompted the
hypothesis that the secreted pentameric IgM may be assembled
randomly from these two populations of subunits. Thus, as
an indirect test of this hypothesis, an experiment was
undertaken to attempt fractionating the secreted 19S
molecules into subpopulations with differing numbers of
hapten binding sites. Specifically purified protein 14PAF
was reabsorbed to an affinity column and sequentially eluted
with increasing (arbitrary) amounts of DNP-OH. The eluted
components were freed of DNP-OH, quantified for protein
and studied by equilibrium dialysis. The results, depicted
in Figure 3, indicate that the initial pentameric IgM anti
body population was composed of subpopulations with differ
ent numbers of binding sites: While this experiment was not
"fine tuned" to the point where it is possible to clearly
say that the pentamers were assembled in a random fashion
from the two forms of subunits, the results are certainly
suggestive.


Figure 2. Equilibrium dialysis with DNP-e-aminocaproate
of unfractionated ( ), and active (oo),
and inactive ( ) 7S subunits from 14PAF.


r/c x 10'6
no
o


Figure 3. Step-wise elutions of protein 14PAF from DNP-lys-
sepharose affinity column using four concentra
tions of hapten. 18% of the eluted protein was
recovered using 3 x 10-4m hapten; 39% using
1 x 103m; 30% using 3 x 10"3m, and 13% using
1 x 10 M.




23
In light of the above finding of two apparently distinct
subpopulations of subunits, the possibility of intramole
cular heterogeneity at the structural level was considered.
The first approach to this question involved subjecting the
mildly reduced and alkylated L chains from each subunit to
SDS polyacrylamide gel electrophoresis without additional
reduction. The banding patterns of these mildly reduced
chains were clearly different in that the L chains from
the active subunits exhibited a somewhat slower mobility
than those from the inactive subunits (see Figure 6 for
example); extensive reduction of each of these chains
resulted in mobilities expected of ^ 22,000 dalton peptides.
The second approach involved subjecting extensively reduced
and alkylated light chains isolated from the unfractionated
subunits and the two fractionated subunit subpopulations
to alkaline-urea gel electrophoresis. The gel patterns of
the L chains from the two subpopulations were clearly
distinct (Figure 4). These suggestions of L chain differ
ences between the two forms of subunits prompted limited
amino acid sequence studies. The results of these studies
(Table 1) indicated several important points. Firstly, the
L chains from the unfractionated parent molecules exhibited
considerable primary structural heterogeneity with two
different amino acids being demonstrable at 5 (and possibly
7) different positions in the first 12 residues. Secondly,
the L chains from the active and inactive reductive subunits
were each homogeneous (through 12 residues) but quite


Figure 4. Alkaline urea polyacrylamide gel electrophoresis
of isolated light chains from unfractionated
(A), inactive (B), and active (C) 7 S subunits.




26
TABLE 1
Amino Terminal Sequences of Light Chains from
Unfractionated 19S and Active and Inactive
7S Reductive Subunits from 14 PAF
7S subunits
Position
19S unfractionated
Active
Inactive
MOPC 21
1
Asp Asn
Asp
As n
Asn
2
Val He
Va 1
He
He
3
Val
Val
Val
Val
4
Met
Met
Met
Met
5
Thr
Thr
Thr
Thr
6
G1 n
G1 n
G1 n
Gin
7
Thr*
Thr*
Ser
Ser
8
Thr Pro
Thr
Pro
Pro
9
Leu
Leu
Lys
Lys
10
Ser
Ser
Ser
Ser
1 1
Leu Met
Leu
Met
Met
1 2
Thr*
Thr*
Ser
Ser
The amino
terminal sequence of
the light
chains from
M0PC-21
(Svasti and Milstein, 1971) is shown for comparison.
*Ser could be masked


27
different from each other, A comparison of the amino termi
nal sequences of the L chains from these two subpopulations
of subunits with the L chains from MOPC-21 (the L chain
secreted by the myeloma cell line used in the original
fusion) indicated that those from the inactive subunits were
identical (through 12 residues) to those from MOPC-21, The
L chains from the active subunits were quite different.
Hence it seems quite likely that the latter L chains were
encoded for by the genome of the plasma cell used in the
fusion. Therefore the secreted pentameric IgM product of
the hybrid cell contained both MOPC-21 and "anti-DNP" L
chains.
The u chains from both the active and inactive subunits
from protein 14PAF were blocked at the amino terminus
(presumably due to a pyrollidone carboxylic acid) and thus
were not amenable to automated sequence analysis. In an
attempt to ascertain if these p chains were functionally
identical, recombinant non-covalent subunits were produced
between all possible equimolar combinations of separated
mildly reduced and alkylated p and L chains derived from both
types of subunits. Each of these recombinants was gqT
filtered and the 'v 1 80,000 dalton material (representing
>90% of the total) subjected to affinity chromatography
on DNP-lys-sepharose. Greater than 95% of the recombinants
formed with L chains from active subunits and p chains from
either active or inactive subunits were observed to absorb
to (and to subsequently hapten elute from) the affinity


28
columns; less than 5% of either of the recombinants formed
with L chains from inactive subunits absorbed to the columns.
Equilibrium dialysis with these latter inactive recombinants
indicated no detectable combining sites for the DNP group;
similar studies had indicated that the isolated y or L
chains bound no detectable hapten (data not shown). On the
other hand, the active recombinants recovered by hapten
elution from immunoadsorbant columns were each observed,
in duplicate experiments, to have an average of about one
combining site with an affinity identical to that of the
parent molecule (Figure 5). Similarly a single study was
performed utilizing halfmers (covalently linked H-L's) from
active subunits obtained from the leading edge of the H
chain peak on the 5 M guanidine Agarose A5M column. After
removal of the guanidine by dialysis against Tris buffer,
these subunits ('v 180,000 dalton material by gel filtration)
displayed an average of one combining site (identical to
Figure 5). To further prove the functional identity of the
y chains present in the original 19S molecules, mildly
reduced and alkylated y chains from either the active or
inactive reductive subunits were recombined with an equimolar
amount of mildly reduced and alkylated L chains derived from
the original molecule (i.e., %60% "anti-DNP" and ^ 40%
MOPC-21 L chains). Each of these recombinants was then
gel filtered on Sephadex G-200 and the 2H-2L chain recombi
nants (representing >90% of the material) were subjected to
affinity chromatography on DNP-lys-sepharose columns;


Figure 5.
Equilibrium dialysis with DNP-e-aminocaproate of
14PAF recombinants using either heavy chains from
active subunits (AH) or inactive subunits (IH)
and light chains from active subunits (AL).
Recombinants made using either heavy chain with
light chains from inactive subunits showed no
detectable binding.


30


31
approximately 40% of the applied material in each case
failed to absorb to the column whereas the remainder ab
sorbed and was recovered by hapten elution. The findings
from this experiment were a) the recombinant subunits which
absorbed to the affinity column contained an average of one
binding site identical in affinity to that seen in the
previous active recombinants (Figure 5) and the original
molecules, and b) the active recombinants contained only
L chains with an SDS polyacrylamide gel electrophoretic
mobility characteristic of the "anti-DNP" L chains whereas
the inactive recombinants appeared to contain only M0PC-21
L chains (Figure 6). Soft laser scans of these stained gels
clearly indicated each of the recombinants to be composed
of equimolar u and L chains. In a further attempt to
ascertain if 14PAF p chains preferentially form recombi
nants with either of the two forms of L chains, mildly
reduced and alkylated chains from active reductive subunits
were mixed with a two-fold molar excess of 14PAF L chains
(consisting of a 60:40 ratio of "anti-DNP" and MOPC-21 L
chains). The resultant mixture was, without subsequent gel
filtration, then subjected to affinity chromatography on a
DNP-lys-sepharose column; approximately 52% of the material
failed to absorb to the column whereas the remaining 48%
absorbed and was recovered by hapten elution. Again, as in
the above case where equimolar p and L chains were mixed,
the active recombinants contained equimolar p chains and
"anti-DNP" L chains when examined by SDS gel electrophoresis.


Figure 6. SDS-PAGE of recombinants formed using heavy
chains from either active or inactive subunits
combined with an equimolar amount of light
chains consisting of 60% from active subunits
and 40% from inactive subunits. Lane A repre
sents unfractionated recombinants; Lane B repre
sents recombinants which absorbed to and were
subsequently hapten eluted from a DNP-lys-
sepharose affinity matrix; and Lane C represents
recombinants which did not absorb to the affinity
column.


33
-.'
w
. V. i
y''5 r
i >' ; w
j:vvS


34
The inactive fraction from this experiment contained y
chains and a greater than three-fold molar excess of L
chains; these L chains were present in a 55:45 ratio of
MOPC-21 to "anti-DNP" L chains. The implications of these
results are considered below.
Discussion
The original purpose of the work undertaken here was
to utilize murine hybridomas as sources of homogeneous IgM
antibodies to test the hypothesis that certain IgM anti
bodies can exhibit intramolecular heterogeneity of ligand
binding that is not attributable to primary structural
differences. Although the presence of two different L
chains in protein 14PAF precludes its utility in this regard,
the studies reported here did reveal several novel and
potentially important aspects of IgM structure and function.
It was not surprising, based upon the observation of "mixed"
molecules secreted by other hybridoma cell lines derived
from Ig secreting myeloma cells (39), that protein 14PAF
contained two different L chains. It was surprising, how
ever, that protein 14PAF was seemingly randomly assembled
from 2y -2L chain subunits that were homogeneous in terms of
L chains. This finding suggests that each type of subunit
may be assembled from y-L chain (or perhaps y-y-L chain)
(40) intermediates which- can only associate with homologous
y-L (or L) chain intermediates to form 2y-2L chain subunits.
The possibility that this putative L chain restriction at
this level is due to the presence of two different y chains


35
is seemingly ruled out by the results of the in vitro
recombination studies. The finding that y chains from
either type of reductive subunit (active or inactive)
could combine with "anti-DNP" L chains to form active
recombinant subunits certainly indicates a high level of
functional similarity in these chains. Similarly, the
observation of homogeneous 2y-2L chain recombinants formed
with a mixture of L chains and y chains from either type
of subunit argues strongly that the intrasubunit L chain
restriction is attributable to the L chains themselves.
In this regard the results obtained from the recombination
experiment using a two-fold molar excess of L chains to y
chains are particularly important in that they show rather
conclusively that either type of L chain can combine equally
well with the y chains. For example if the y chains had a
preference for recombining with the "anti-DNP" L chains,
no y chains would be expected in the inactive recombinant
fraction as they would all be found in the active fraction.
Likewise, if the y chains had shown a preference for the
MOPC-21 chains, >_ 85% of the recombination mixture should
have been found in the inactive recombinant fraction. As
pointed out in the results, 52% of the recombinant mixture
was found in the inactive fraction (hypothetically, one
would predict 53% if assembly was random for each type L
chain). Furthermore, the presence of "anti-DNP" L chains
along with y chains in the inactive mixture demonstrates
that the "anti-DNP" L chains were not a limiting factor in


36
forming recombinants but that the y chains must have an
equal capacity to form inactive recombinants with MOPC-21
L chains or active recombinants with "anti-DNP" L chains,
In addition the presence of ^ 55% MOPC-21 L chains to ^ 45%
"anti-DNP" L chains in the inactive fraction is almost
precisely what would have been expected if assembly of the
recombinants was random for both L chains (hypothetically,
one would predict 57% MOPC-21 L chains and 43% "anti-DNP"
L chains). It thus appears that assembly of 14PAF subunits
i n vitro mimics assembly of subunits i_n vivo both with
respect to ratios of active to inactive subunits and the
homogeneity in terms of L chains. Hence, the conclusion
from this study is that the combination of one of the L
chains with the y chain produced by this cell line results
in an assembly intermediate that will only associate with
a homologous y-L chain pair to form a subunit. Such
restriction may result from different L chain imposed y
chain conformational differences; future work is required
to determine if such putative y chain conformational differ
ences are caused by the seemingly large conformational
differences (as manifested by the differences in SDS gel
electrophoretic mobilities) between mildly reduced and
alkylated MOPC-21 and protein 14PAF "anti-DNP" L chains.
A final point of interest, and potential importance,
from these initial studies with protein 14PAF was the
failure to achieve the anticipated number of combining
sites in the active recombinants. The 2y-2L chain in vitro


recombinants formed with y and L chains (or halfmers)
from active 2y-2L chain reductive subunits should have
exhibited two ligand binding sites if the i_n vitro recom
bination process worked perfectly, i.e., such as seemingly
was the case during intracellular assembly (see Figure 2).
On the other hand, due to the technical intricacies of such
experimental manipulations, a loss of (or failure to
recover) some of the active sites* Pool'd not be unexpected.
Thus, for the sake of discussion, if 50% of the sites were
lost (as suggested by the data in Figure 5) it would be
expected that such losses might be random and hence about
25% of the recombinants should have two sites, 50% have one
site and 25% have no active sites. In such a case, only
75% of the 2y-2L chain recombinants would be expected to
absorb to an affinity matrix; these absorbed and hapten
eluted recombinants should exhibit an average of 1.3 sites
per molecule. The results obtained with 2y-2L chain recom
binants formed with protein 14PAF "anti-DNP" L chains and
either of the two y chains (from active or inactive reduc
tive subunits) clearly showed that >95% of the recombinants
absorbed to a DNP affinity column and therefore had at least
one active site. Furthermore equilibrium dialysis with
these active recombinants indicated an average valence of
only one. Hence the conclusion from these results must be
that each 2y-2L chain recombinant had but one active site
for ligand binding. The reason for this rather striking
result is unknown but it would seem appropriate to speculate


38
that it may be attributable to intrasubunit conformational
differences arising during the i_n vitro assembly; perhaps
one mildly reduced and alkylated y-L chain somehow
influenced the conformation of the other pair to result in
a 2y-2L chain recombinant with but one active site for the
DNP moiety. Such conformational differences, if demon
strable in the i_n vitro recombinants of protein 14PAF,
would certainly justify future studies regarding this
possible explanation for heterogeneity of ligand binding
by other Ig M antibodies.


CHAPTER III
INTRAMOLECULAR HETEROGENEITY OF TWO IgM ANTIBODIES
TO THE DNP MOIETY DERIVED FROM MURINE
HYBRIDOMA CELL LINES
Introduction
As previously discussed the results obtained by
several different laboratories using IgM antibodies from a
wide variety of sources indicated that some,, but not all,
of these antibodies appeared to exhibit intramolecular
heterogeneity of ligand binding. As mentioned, these
earlier studies were limited by the relatively small amounts
and the structural heterogeneity of the IgM molecules avail
able. In order to clearly resolve this question of intra
molecular heterogeneity it has become imperative to
develop approaches for obtaining sufficient amounts of
structurally homogeneous mammalian IgM antibodies with
specificities for defined ligands. Chapter II describes
the initial attempt at obtaining a homogeneous anti-DNP
IgM antibody using hybridoma technology. The study of the
molecule obtained, designated 14PAF, resulted in some rather
interesting and novel observations which might be pertinent
to the question of intramolecular heterogeneity. However,
it was felt that this molecule would not, in reality,
permit an adequate assessment of the hypothesis regarding
intramolecular heterogeneity due to the presence of two
different L chains in the secreted pentamer.
-39-


40
This present chapter describes additional cell fusions
which were undertaken using two myeloma lines, NP3 (41)
and SP2/0 (42), which do not produce immunoglobulin. Two
of the cell lines obtained from these fusions have been
designated NP3-17 Cl-20 and SP2/0 1-64 C1 -12. Each of
these cell lines grows as ascitic tumors in mice and yields
moderate amounts ('v 1 mg/ml) of 19S IgM antibodies to the
DNP moiety. Although apparently structurally homogeneous
(as manifested by alkaline urea gel patterns of isolated
L chains from both proteins and by limited amino acid
sequence analysis of H and L chains from one, NP3-17
Cl-20), the hapten binding data obtained for each of these
proteins indicate an average of only five high affinity
binding sites for the DNP group per 19S molecule. Greater
than 95% of the reductive 7S subunits absorbed to and were
hapten eluted from a DNP-lys-sepharose affinity column.
When examined by equilibrium dialysis, each subunit contained
an average of one high affinity binding site. One experi
ment aimed at defining the molecular basis of this observed
binding heterogeneity attempted to determine if asymmetrical
carbohydrate attachment to the molecule is in any way
involved. Tunicamycin, an antibiotic that prevents glyco-
sylation of glycoproteins, was used in an effort to isolate
carbohydrate-freeIgM antibodies and to study their binding
properties.
Other studies attempting to define the molecular basis
of the observed binding heterogeneity involved physically


41
separating the two types of sites, Mild reduction of each
of these IgM molecules yielded predominantly halfmers (H-L)
in dissociating buffers. Experiments designed to disrupt
the non-covalent associations between opposing halfmers
of a 7S subunit, as well as trypsin digestion of both 7S
molecules, indicated that conformational differences may
exist between the two binding site regions of an individual
7S subunit. A proposed mechanism for this heterogeneity
will be discussed.
Materials and Methods
Hybridomas
Cell fusions were performed as described in Chapter II
using the myeloma cell lines P3-X63-Ag8.653 (41) and
SP2/0-Ag-14 (42). After substantial growth, culture
supernatants were screened for IgM antibodies to the DNP
moiety using a radioimmunoassay (34) or passive hemagglutin
ation with DNP conjugated sheep red blood cells. Cultures
were cloned in soft agarose (35) and individual positive
clones were subsequently injected into BALB/c mice primed
with pristane to obtain ascitic tumors. Two such clones,
designated NP3-17 Cl-20 and SP2/0 1-64 Cl -12, were selected
for the studies reported here.
Immunochemical Procedures
IgM antibodies to the DNP moiety were purified from
ascitic fluid by affinity chromatography, freed of hapten
by Dowex 1X-8 ion exchange chromatography, and examined by
equilibrium dialysis against H-DNP-e-aminocaproate as


42
described previously (18). For calculating protein concen
trations of the pentameric IgM antibodies, a molecular
weight of 900,000 daltons and an extinction coefficient
11
(Eoonm t ) of 11.0 were assumed.
280nm I cm
Initially, reduced 7S subunits of both proteins were
prepared by subjecting purified 19S material (concentra
tions ranged from 3-10 mg/ml) to reduction with .01 M
and .005 M 2-mercaptoethanol for IgM antibodies from
NP3-17 Cl-20 and SP2/0 1-64 Cl -12, respectively. These
levels of 2-ME were determined empirically for each molecule
as that level of reducing agent which yielded the greatest
percentage of subunits containing covalently linked H and
L chains or halfmers (covalently linked 2H-2L chain 7S
subunits could not be obtained; Figure 7). These reduc
tions were performed in 0.5 M Tris-HCl, pH 8.0 for one
hour at 22C followed by alkylation with 0.15 M iodoace-
tamide for one hour on ice. Gel filtration under non
denaturing conditions (Sephadex G-200 equilibrated with
0.15 M NaCl .01 M Tris-HCl, pH 7.4) indicated that ^ 95%
of such reduced and alkylated 7S material eluted in a
volume expected to contain 180,000 dalton proteins;
analysis under denaturing conditions without additional
reduction indicated the 7S subunits dissociated predomi
nantly into halfmers (H-L molecules). Subsequently it was
found that halfmers could be obtained in non-denaturing
buffers by mild reduction of dilute protein solutions
(.5-1.0 mg/ml) in .5 M Tris-HCl pH 8.5.


Figure 7.
Mild reduction profile of 19S IgM from hybridoma
SP2/0 1-64 Cl-12 using increasing amounts of
2 ME from left to right. Lane 1, ,001 M;
Lane 2, .005 M; Lane 3, .01 M; Lane 4, .05 M;
Lane 5, .1 M. Profiles of IgM from NP3-17 Cl-20
were similar with .01 M 2 ME being optimum for
the production of halfmers (H-L).


44
12 3 4 5
i9s m


45
Alkaline-urea gel electrophoresis of extensively
reduced L chains was performed as in the preceding chapter
by the method of Reisfield and Small (36). SDS-polyacryla-
mide gel electrophoresis was performed according to the
method of Laemmli (37).
Amino terminal sequence studies were performed by
Edman degradation using an automated Beckman sequenator.
PTH derivatives were identified by high pressure liquid
chromatography (38).
Tryptic hydrolysis of 7S reductive subunits (5-10
mg/ml) was performed for various time periods following
the protocol of Klapper et al. (43). Briefly a 1:100
enzyme (trypsin, 3-X crystallized, Worthington Biochemical
Corp.) to substrate ratio was established and the tempera
ture was maintained at 37C throughout the digestion.
Trypsinization was done in .25 MTris-HCl, pH 8.3, and
.01 M CaC12 was added to the reaction mixture prior to
adding trypsin (44). Tryptic digestions were halted by the
addition of equimolar amounts of soybean trypsin inhibitor
(45). Faby fragments were isolated by gel filtration (G-200
Sephadex equilibrated with 0.15 M NaCl, .01 M Tris-HCl,
pH 7.4), Active Fab's were then selected using a DNP-
lysine-sepharose immunoadsorbant. Equilibrium dialysis was
performed on these Fab's as previously described in this
text. A molecular weight of 50,000 and an extinction coef
ficient of 13.0 were used to calculate Fab concentrations
(44).


46
Attempts were made to dissociate putative active
halfmers by disturbing non-covalent interactions between
opposing y chains. Mildly reduced antibody was allowed
to absorb to a DNP-lys-sepharose affinity column followed
by exposure of the column to chaotropic or ionic dissociating
agents. Various concentrations of the following agents
were used: guanidine HC1, sodium thiocyanate, acetate
buffer (pH 5.0), isopropyl alcohol, NP-40, arid Tris-HCl
(pH 8.5).
Affinity labeling of reductive subunits from SP2/0
1-64 C1 -12 was attempted to irreversibly block the active
anti-DNP site on each 7S monomer without affecting the
inactive site. A two-fold excess of dinitrof1uorobenzene
(DNFB) and dinitrobenzenesulfonate (DNBS) to antibody was
used. Two milliliters of a 7.2 x 10~^ M concentration of
antibody in .1 M borate buffer, pH 8.3 was allowed to react
for two hours at room temperature with either DNFB or DNBS
_5
at concentrations of 1.44 x 10 M. After two hours an
equal volume of .1 M Tris-HCl, pH 8.0, was added to each
tube to stop the labeling reaction. The antibody was
dialyzed against Tris-buffered saline (.15 M NaCl .01 M
Tris-HCl, pH 7.4) overnight. The treated antibody was
passed over a DNP-lys-sepharose affinity column and any non
absorbing antibody was measured spectrophotometrically at
280nm. It was hoped that when such treated subunits were
placed in 2.0 M guanidine-HCl and allowed to "refold" upon


47
dialysis against Tris-buffered saline that 50% of the
original inactive sites would become active.
Attempts were made to obtain an anti-idiotypic anti
serum to IgM from SP2/0 1-64 Cl-12. It was hoped that such
antiserum might detect idiotypic differences between mole
cules which could bind hapten and those which could not.
Each of three BAL B/c mice was initially immunized intra-
4 '
peritoneally with 100 pg of 19S antibody from SP/20 1-64
Cl-12 emulsified in complete Freund's adjuvant. Each
mouse received three additional intraperitoneal injections
of protein without adjuvant over the next two months. One
month into the immunization schedule each mouse received
g
1 x 10 SP2/0.14 myeloma cells in order to produce an
ascitic tumor. Three days after the end of the last
immunization ascitic fluid was removed and assayed for
anti idiotypic antibodies.
Tunicamycin Experiment
Cells. Six x 10^ SP2/0 1-64 Cl-12 cells were incubated
in 10 ml of methionine-free Dulbecco's MEM (with L-gluta-
mine, penicillin and streptomycin) with 15% fetal calf
serum. The experiments were run in a humidified atmosphere
of 5% C02:95% air at 37C.
35
Cell labeling. Cells were labeled with S-methionine
(American Searle, Arlington Heights, IL, 900 Ci/mmole) by
the addition of 100 pCi to 10 ml cultures. When tunicamycin
(Tm) (Eli Lilly, Co., Indianapolis, IN) was employed, cells


48
were suspended in the above medium containing 10 yg/ml
Tm for one hour before addition of the label. Cells were
labeled for eight hours before supernatants were removed
for analysis.
Purification of antigen-specific counts. Cells were
removed from supernatants by centrifugation at 400g for
15 minutes. Cell-free supernatants were passed over a
DNP-1ys-sepharose affinity column to purify antigen-
specific radioactive molecules. This purified antibody
was hapten eluted (.1 M DNP-OH) and subjected to Dowex
1X8 ion exchange chromatography to remove the DNP-OH.
It was necessary at this point to spike the radioactive
IgM with unlabeled IgM in order to avoid significant loss
of radioactivity on the Dowex column. Radioactivity was
assessed by adding 10 y 1 of sample to 2 ml of scintillation
cocktail containing 50% (v/v) Triton-X-100.
Gel analysis. Tm-treated and non-treated purified
antibodies were subjected to SDS-DATD-acrylamide gel chroma
tography (46). Gels were dried, overlaid with Kodak X-OMat
AR film and stored between intensifying screens for desired
exposure times.
Sequential hapten elutions. Purified Tm- or non-Tm-
treated antibody was mildly reduced with .1 M 2ME for one
hour and alkylated (.15 M iodoacetamide) for one hour on
ice. These reductive subunits were allowed to absorb to a
small DNP-lys-sepharose column and subjected to step-wise
gradient hapten elutions (beginning with 1 X 10 ^ M DNP-
OH) until all radioactivity was eluted.


49
Analysis of nonspecific radioactivity from supernatants.
Radioactivity from Tm- or non-Tm-treated supernatants was
examined for nonspecific IgM by immunoprecipitating super
natant fluid which had been freed of specific antibody by
affinity chromatography. Twenty microliters of a rabbit
anti-mouse y chain antiserum was added to .1 ml of anti-DNP
free supernatant and incubated for one hour on ice. This
was followed by addition of .2 ml of a 50% suspension of
Staphylococcus A (Cowan I strain) and incubated for 15
minutes on ice. This mixture was centrifuged for three
minutes at 15,000 rpm. Pellets were resuspended in 1%
SDS-Tris buffered saline and samples were removed for
scintillation counting.
Analysis of cell lysates. Pelleted cells from Tm-
or non-Tm-treated cultures were resuspended in 4 ml of
.1% NP40 in .04 M Tris-buffered saline with .01 M EDTA,
pH 8.3. Cell debris was removed by centrifugation at
500g for twenty minutes. Supernatants were then freed
of DNP specific antibodies by affinity chromatography.
Both purified antibody and antibody-free supernatants were
assayed by the methods described above.
Results
Functional and Structural Characterization of the IgM
Antibodies
The hybridoma lines utilized here (NP3-17 Cl-20 and
SP2/0 1-64 C1 -12) grow as ascitic tumors in BALB/c mice
and yield moderate amounts 1 mg/ml) of 19S IgM antibodies
to the DNP moiety. Equilibrium dialysis studies using the


50
hapten DNP-eaminocaproate indicate each to contain an
average of five high affinity (Ka^lO^M ^) binding sites
(Figure 8). Furthermore, ^ 95% of the reductive 7S
subunits of each of these proteins absorb to and subse
quently can be hapten eluted from a DNP-lys-sepharose
affinity column. These subunits when examined by equili
brium dialysis again using DNP-e-aminocaproate indicate
each to contain an average of one high affinity binding
site. Structural features of these two proteins indicate
a considerable degree of homogeneity as might be antici
pated using non-producing myeloma cell lines as the parent
line for the fusion process. Alkaline-urea gel electro
phoresis of the isolated L chains from each protein revealed
banding patterns indicating considerably restricted hetero
geneity (Figure 9). In addition, limited amino terminal
sequence analysis of the H and L chains from the NP3-17
Cl-20 protein indicate each to be homogeneous (Table 2).
The H and L chains from SP2/0 1-64 C1 -12 IgM were not
amenable to sequence analysis (presumably due to blocked
amino terminii; pyrollidone carboxylic acid).
In experiments designed to measure the binding ability
of either of these 19S molecules after treatment with 5.0 M
guanidine-HCl for one hour followed by dialysis against
Tris buffer, it was found by equilibrium dialysis that the
high affinity binding sites were totally destroyed; no low
affinity sites could be detected (data not shown).


Figure 8. Equilibrium dialysis of 19S IgM from hybridoma
SP2/0 1-64 Cl-12 with DNP-e-aminocaproate.
Results for NP3-17 Cl-20 were similar.


52


Figure 9. Alkaline urea polyacrylamide gel electrophores
of isolated light chains from (A) SP2/0 1-64
Cl-12 and (B) NP3-17 Cl-20.


54


55
TABLE 2
Amino Terminal Sequences of Heavy and Light
Chains of IgM Antibody to DNP from a Hybri-
dome Line NP3-17 CL-20
RESIDUE
H CHAIN
L CHAIN
1
Glu
Glu
2
Val
As n
3
G1 n
Val
4
Leu
Leu
5
Gin
Thr
6
Gin
G1 n
7
Ser
Ser
8
Gin
Pro
9
P ro
A1 a
1 0
Glu
He
1 1
Leu
Met
12
Val
Ser


56
Separation of Halfmers (H-L)
Mild reduction of either of these IgM molecules
resulted in the formation of noncovalently associated
halfmers (H-L) in nondissociating buffers. These mole
cules gel filtered on G-200 Sephadex as would be expected
of a 7S, 180,000 dalton protein. However on SDS-PAGE
(10% acrylamide) without further reduction these molecules
migrated as ^90,000 dalton proteins, i.e., halfmers. Upon
subsequent extensive reduction in SDS the ^90,000 dalton
material dissociated into equimolar 'WO.OOO dalton H chains
and ^22,000 dalton L chains. Since these halfmers non
covalently associated in nondissociating buffers it was felt
that appropriate denaturing or chaotropic agents might be
able to disturb the noncovalent interactions without
destroying the binding site of the "good" halfmers, there
fore allowing elution and recovery of the "bad" halfmers.
Using a "grab bag" approach, various concentrations of the
following agents were used: guanidine-HCl sodium thio
cyanate, acetate buffer (pH 5.0), isopropyl alcohol, NP-40,
and Tris-HCl (pH 8.5). Initially it was observed that two
column volumes of 2.0 M guanidine-HCl would elute 50% of
the "7S" antibody from the DNP-lys-sepharose column with
the remaining 50% being eluted with .1 M DNP-0H. After
dialysis against Tris buffer the guanidine-eluted fraction
would subsequently bind to the DNP affinity matrix.
Furthermore both the guanidine-eluted and the DNP-eluted
material exhibited one binding site per 2H-2L chain


57
subunit. The major problem in interpreting this experi
ment was the observation that all of the bound antibody
could be eluted if more than two column volumes of 2.0 M
guanidine-HCl was used. Therefore there are two distinctly
different interpretations of the data: 1) It is possible
that the two column volumes of guanidine-HCl were eluting
preferentially the "bad" halfmers from the affinity column
and that any more than two volumes began to denature and
elute "good" halfmers. This liberal interpretation of the
data implies that if "bad" halfmefs were eluted by the
smaller amount of guanidine-HCl, half of them must have
subsequently become "good" haTfmerS when dialyzed into Tris
buffer. Proof of this interpretation would substantiate the
theory of conformational differences between opposing
binding sites of a "7S" molecule. 2) The more conservative
interpretation (and more likely) of the data would be that
the guanidine-HCl elution was not preferential but rather
nonspecifically eluted 50% of the antibody. All but one of
the other dissociating agents tried either eluted all of
the antibody from the column or was not able to elute any
of the antibody. Only recently it was observed that .5 M
Tris-HCl (pH 8.5) could elute 50% of the "7S" antibody
absorbed to the DNP affinity column. The use of .5 M
Tris-HCl was prompted by an observation which warrants some
discussion at this point. Rather serendipitously, it was
observed that mild reduction in .5 M Tris-HCl of either of
the two 19S molecules at dilute protein concentrations


58
(<1 mg/ml) yielded subunits of which 50% would bind to a
DNP affinity matrix (active) and 50% would not (inactive)
providing the protein was left undiluted as applied to the
affinity column. If the reduced mixture was first concen
trated ^90% of the antibody would bind to the column.
When examined by equilibrium dialysis the unconcentrated
inactive fraction displayed no binding sites, whereas the
active fraction displayed an average of one site per pre
sumed 2H-2L chain 7S subunit (Figure 10). Interestingly,
if the inactive fraction was first concentrated to > 1 mg/ml
a small percentage (.3 sites/2H-2L chain subunit) of high
affinity sites could be generated. SDS-acrylamide gel
electrophoresis of each fraction demonstrated that these
molecules were predominantly halfmers (H-L) in dissociating
buffer (Figure 11).
Hapten Binding by Tryptic Fragments
The finding of only one active hapten binding site in
each reductive subunit from the two hybridoma-derived IgM
antibodies prompted studies aimed at separating "good"
(hapten binding) from "bad" (no hapten binding) Fab frag
ments. The strategy employed for this purpose involved
attempts at preparing such Fab's by trypsinizing reductive
subunits. Originally each reductive molecule was subjected
to trypsinization for various times and aliquots were
analyzed by SDS-PAGE (Figure 12). It was observed that
although relatively little digestion occurred within the
first two hours, digestion to Fa by 1 s (defined as ^50,000


Figure 10. Equilibrium dialysis using DNP-e-aminocaproate
of NP3-17 Cl-20 "7S" active ( ) andinactive
(o O) fractions resulting from mild reduction
at dilute protein concentrations (< 1 mg/ml).
The unfractionated starting material was iden
tical to the active fraction. Concentration of
the inactive fraction () resulted in the
generation of some sites.


o
VO
o
cJ
o
c\j
oex


Figure 11. 10% polyacrylamide gel electrophoresis (SDS)
of NP3-17 Cl-20 "7 S". Lane A, unfractionated;
Lane B, active fraction, and Lane C, inactive
fraction. D, E, and F depict the SDS-PAGE
patterns of samples A, B, and C after extensive
reduction .


62


Figure 12. 10% polyacrylamide gel electrophoresis of timed
trypsin digestions of SP2/0 1-64 Cl-12 7S
reductive subunits. Length of exposure to
trypsin is designated in hours above each
sample. Panel A depicts unreduced samples;
Panel B depicts the same samples upon reduction
with .1 M 2 ME in 1% SDS in a boiling H^O bath
for three minutes.


64
A
O I 2 3 4 5 24
H-L
a
B
O I 2 3 4 5 24
H
L


65
dalton material containing L chains and part of the y
chains) was fairly rapid between 3-5 hours; very little
change was seen over the course of the subsequent 19 hours.
In addition, no F(ab ) ^ s were obtained since the inter-H
chain disulfides were cleaved by the mild reduction step
used in preparing the subunits. Large scale digestions
were performed for periods longer than three hours (five
hours for SP2/0 1-64 Cl-12 and 18 hours for NP3-17 Cl-20) .
Very little difference, if any, could be detected in their
digestion profiles (G-200 and SDS-PAGE). The results of
these large scale digestions are represented in Figures
13 and 14. Surprisingly, only 30-35% of the total protein
(^50% of the expected yield) was recovered as Fab's when
the digested material was chromatographed on G-200 Sephadex
equilibrated in Tris buffer. The remainder of the material
was seen as small peptides. However, as demonstrated in
Figure 15, the Fab's which were recovered displayed one
high affinity binding site as measured by equilibrium dialy
sis. An important finding was that those Fab's containing
one site accounted for ^90% of the binding sites present
in the untrypsinized starting material. Thus the digested
Fab's seemingly were exclusively of the type that contained
no active sites for binding the DNP ligand.
Asymmetry of Carbohydrate
One possible explanation for heterogeneity of binding
within the pentamer and reductive subunits of these molecules
could be the presence of an asymmetrical attachment of


Figure 13. Sephadex G-200 elution profile of SP2/0 1-64
Cl-12 Fab's obtained after trypsinization for
five hours. An eighteen hour trypsinization
of NP3-17 Cl-20 produced a similar profile.


67
7S
(2 ml f factions)


Figure 14.
10% polyacrylamide gel electrophoresis (SOS)
of unfractionated (A), and active (C) Fab's
obtained from five hour trypsinization of
SP2/0 1-64 Cl-12; (B) contains inactive, non-
Fab material. D, E, and F depict the SDS-PAGE
patterns of samples A, B, and C after extensive
reduction. SDS-PAGE patterns of NP3-17 Cl-20
Fab's were indistinguishable.


6 9
ABC D E F


Figure 15. Equilibrium dialysis of Fab fragments obtained
from a five hour trypsinization of SP2/0 1-64
Cl-12 with DNP-e-aminocaproate. Results from
equilibrium dialysis with Fab's from NP3-17
Cl-20 were identical.




72
carbohydrate (see 47). This possibility prompted an exper
iment designed to obtain carbohydrate-free IgM antibodies.
Tunicamycin (Tm), an antibiotic that prevents glycosylation
of glycoproteins by blocking the formation of M-acetyl-
glucosamine-lipid intermediates (48), was used to block
glycosylation of the IgM produced by SP2/0 1-64 Cl-12. It
35
was found that culture supernatants from S-methionine
k T *
labeled cells treated for eight hours with Tm contained only
1% (15,000 cpm) of the antibody level (1.5 x 10^ cpm) found
35
in supernatants from non-Tm treated S-methionine labeled
cells. Furthermore, the IgM that was purified from the
supernatants of Tm-treated cells appeared to be no differ
ent from IgM purified from the supernatants of non-Tm
treated cells. This was evident by an autoradiogram of
purified antibody run on an SDS-DATD acrylamide gel (Figure
16) and by sequential hapten elutions of reductive 7S
subunits (Table 3). Radioactive material which was not
specifically purified by an affinity column was shown by
immunoprecipitation to be free of IgM (Table 4). Attempts
were made to isolate carbohydrate-free antibody from cell
lysates of Tm-treated' cells, but unfortunately, none was
detected, It is not known if lower levels of Tm or other
incubation times might be advantageous for the production
of unglycosylated IgM by this cell line.
Anti-idiotypic Antiserum
In terms of assessing the variability of antibody sites
the utilization of anti-idiotypic antibodies has become a


3 5
Figure 16. Autoradiogram of specifically purified S-
methionine molecules from supernatants or
cell lysates of Tm- or non-Tm-treated SP2/0
1-64 Cl-12 cells.
Lane A and H Purified antibody from non-Tm-
treated cultures;
Lane B and G Purified antibody from Tm-
treated cultures ;
Lane C and F Purified antibody from non-Tm-
treated cell lysates;
Lane D and E Purified antibody from Tm-treated-
cell lysates.
Samples E H were reduced with 1 M 2 ME and
boiled in 1% SDS for three minutes.


74


TABLE 3
Comparison of Hapten Elution Profiles of
Mildly Reduced SP2/0 1-64 Cl -12 IgM Antibody
from Tm- and Non-Tm-treated Culture Supernatants
Antibody from Antibody from ?
Non-Tm-Treated Culture Tm-Treated Culture^
Supernatants Supernatants
(DNP-OH)
Hapten Concentration CPM Eluted 0D280 E1uted3 CPM Eluted 0D280 E1uted
(%
of Total)
(% of Total)
5 x 10"5
M
0
0
0
0
1 x 10"4
M
4200
(10.5)
.275
650
(8.0)
.210
5x10
M
7500
08.8)
.267
1000
(12.5)
.270
1 x 10_J
M
1 7000
(43,0)
.328
3600
(45.0)
.342
5 x 10J
M
4800
(12.0)
. 264
650
(8.0)
. 1 80
1 x 10
M
0
0.
0
0
34000
(85,0)
1 .1
5800
(.73,0)
1 .002
I nactive
Fraction
4000
(10.0)
,2
1 500
(19.0)
.18
Total
38000
(95,0)
1 .3
7300
(94.0)
1.182
^Loaded 1 ml DNP-lys-sepharose affinity column with 40,000 cpm.
2
Loaded 1 ml DNP-lys-sepharose affinity column with 8,000 cpm.
3
.5 ml of non-radioactive SP2/0 1-64 Cl-12 antibody (O.D.28Q = 2.0) was added
to each radioactive antibody preparation prior to reduction for use as an optical
density marker.


TABLE 4
Immunoprecipitation of Tunicamycin (Tm)-treated and
Non-Tm-treated Culture Supernatants and Cell Lysates
Amount of Rabbit % of Radioactivity
Sample anti-mouse y chain (ul) Precipitated
I.Control Antibody^ 20 100B
40 100
100 100
0 (100 ul Normal 2
Rabbit Serum)
II.Culture medium spiked with Control Antibody 100 100
r
III.Inactive fraction from non-Tm-treated cultures 100 .004
IV. Inactive fraction from non-Tm-treated culture supernatants 0 (100 ul Normal .008
Rabbit Serum)
V. Inactive fraction from Tm-treated culture supernatants 100 .004
VI. Inactive fraction from non-Tm-treated cell lysates 100 .005
VII. Inactive fraction from Tm-treated cell lysates 100 .004
35
A S-labeled affinity purified antibody from non-Tm-treated culture supernatants.
B Percentages were normalized to 100% to account for a uniform loss of radioactive material throughout
the experiment due to non-specific stickiness.
C Radioactivity which was not absorbed by the DNP-lys-sepharose affinity column (i.e. antibody to DNP
removed).
O'


77
powerful tool (49). The purpose of developing such an
antiserum in this study was hopefully to allow detection
of idiotypic differences between "good" and "bad" halfmers.
Unfortunately no anti-idiotypic antibodies could be detected
in the ascitic fluids of these animals by Ouchterlony
analysis against the intact 19S molecule. Perhaps a better
approach might be to immunize guinea pigs and absorb this
antiserum with the necessary mouse proteins until desired
idiotypic specificity is achieved.
Affinity Labeling
Affinity labeling of various anti-DNP antibodies has
been successfully utilized to either completely or partially
block the antigen combining site via covalently coupling
antigen in the site (50). This approach was.attempted with
hopes of obtaining "7S" subunits with inactive sites. By
placing these inactive subunits in 2.0 M guanidine-HCl and
subsequent dialysis against Tris-buffered saline the gene
ration of active sites might be possible. This result
would argue that inactive sites have the capability of
binding to antigen but when paired with an active site
assume the wrong conformation for antigen binding. Unfor
tunately in this experiment conditions were not appropriate
for affinity labeling as no inactive antibody passed through
the affinity column. It is quite possible that the appro
priate conditions for affinity labeling could be achieved
and should be pursued in future studies.


78
Discussion
The purpose of the work described in Chapter III was
to utilize murine hybridomas as sources of homogeneous IgM
antibodies to test the hypothesis that certain IgM anti
bodies can exhibit intramolecular heterogeneity of ligand
binding that is not attributable to primary structural
differences. The results of polyethylene glycol induced
fusions using non-immunoglobulin producing cell lines
indicated that stable, antigen specific, IgM secreting
cell lines can be obtained. The two IgM antibodies
obtained from the hybridomas displayed an unusually high
affinity ('v lO^M-"*) for the DNP hapten. It is not known
if cells producing lower affinity IgM antibodies (10 -10 M )
were present as a result of the fusion process and were
missed by the screening procedure used or if, for one
reason or another, cells producing such antibodies simply
did not exist. Nevertheless, these antibodies did meet the
necessary criteria for testing the original hypothesis in
that 1) they were specifically induced pentameric antibodies
which displayed binding heterogeneity, i.e., only five DNP
binding sites. This heterogeneity appeared to be of an
intramolecul ar nature as opposed to intermolecular hetero
geneity based upon the following premises. It seems rather
unlikely, due to the nature of hybridoma derived antibodies,
that the two hybridoma cell lines derived (NP3-17 C1 -20 and
SP2/0 1-64 C1 -12) from the two different myeloma cell lines
used (NP3 and SP2/0) could both manufacture and secrete


79
in equal amounts two nondistinct populations of IgM anti
bodies, one of which had ten binding sites for DNP and one
of which had no binding sites for DNP, Furthermore,
essentially all (> 90%) of the "7S" re.ductiye. subunits
absorbed to (and subsequently hapten eluted from) a DNP-
lys-sepharose column. These subunits when measured by
equilibrium dialysis displayed an average of one site per
7S subunit. These results clearly support the interpreta
tion that each 7S subunit must contain only one site, i.e.,
there were none which contained two and likewise there
were none which displayed no DNP-binding sites. 2) These
IgM antibodies also met the criterion of being structurally
homogeneous, at least by the methods employed. Comparison
of alkaline urea gel banding patterns of myeloma protein
L chains with the banding patterns of these hybridoma L
chains demonstrated considerable structural homogeneity
(for example, compare with the banding pattern of MOPC-21
L chain, Figure 4, Chapter II). Likewise limited amino
acid sequence analysis certainly indicated homogeneity
with NP3-17 Cl 2 0. Undoubtedly, complete amino acid
sequences of both proteins or sequence analysis at least
through the first hypervariable region would be invaluable
in this regard. However, the question becomes more perti
nent when putative inactive sites are separated from active
sites. Perhaps at that stage additional sequence data, as
well as peptide mapping, isoelectric focusing, and/or idio
typic analysis would be beneficial.


80
The results from mild reduction of both IgM antibodies
indicated that the 19S molecules proceeded directly from
the 19S covalent pentameric form to covalent halfmers (H-L).
For example Figure 7 demonstrates that a concentration of
.001 M 2ME does relatively little to change the covalent
structure of the molecule whereas a concentration of .005
M 2ME has converted all of the heavy molecular weight
molecules predominantly to halfmers. The obvious inter
pretation of this finding is that for these molecules the
inter-heavy chain disulfides are as susceptible to reduction
as are the inter-subunit disulfides. This fact could
become important in uncovering a possible mechanism for
intramolecular heterogeneity. According to Askonas and
Parkhouse (51, p. 632) the intracellular assembly pathway
for IgM "mirrors the resistance of interchain disulphide
bonds to reduction." If this is true with these hybridoma-
derived molecules, it would appear that these molecules are
assembled through a H-L chain intermediate based upon their
reduction profiles. If assembly of IgM is involved in the
observed heterogeneity, comparisons of reduction profiles
of these IgM antibodies with reduction profiles of other
IgM antibodies which possess ten homogeneous binding sites
might bring to light an assembly intermediate (for example
H-H-L, see 40) responsible for the phenomenon.
Examination of the results from experiments designed to
separate noncova1ently associated halfmers yielded few clear
interpretations. This was in part due to the fact that the


81
amount of dissociating agent used (two column yolumes of
2.0 M guanidine-HCl) was critical in removing 50% of the
antibody absorbed on the affinity column. Any more than
this amount or subsequent elutions with the same amount
eluted more antibody from the column. As pointed out
previously the most likely interpretation of these results
would be that the guanidine-HCl elution was not preferen
tial but rather nonspec ifi cal 1y eluted 50% of the antibody.
Likewise, results obtained when .5 M Tris-HCl was used to
dissociate the halfmers were equally difficult to interpret
due to the problems associated with total recovery of hapten
binding sites. Neither the 50% fraction which was eluted
from the affinity column by .5 M Tris-HCl nor the 50%
fraction which passed through the DNP-immunoabsorbant when
the mild reduction was performed on dilute solutions of
antibody, displayed any active binding sites by equilibrium
dialysis. The hapten eluted fractions (the remaining 50%)
in both cases displayed only one active site per 2H-2L
chain subunit, thus accounting for only one half of the
original sites in the unfractionated subunit population.
Although equilibrium constants were not measured for the
noncovalent association of halfmers used in this study,
one published report of halfmer association using three
other IgM monoclonal antibodies gives an equilibrium constant
of 2.3 X 10 ^ moles halfmers ^ for the reaction one halfmer
two halfmers (52). It is quite possible that equili
brium constants of this magnitude or higher may exist for


82
the hybridoma-derived IgM antibodies described in this
study. If so, separation of the putatiye "good and bad
halfmers" using chaotropic or dissociating agents may be
impossible, as these agents may equally disrupt hapten
binding and halfmer association. Unlike the previous
study mentioned on halfmer association Parkhouse (53*, p. 640)
in describing halfmer association with IgM from the mouse
myeloma MOPC 104E states:
Since, therefore, there is no pronounced
tendency for non-covalent interaction between
HL subunits it is no surprise to find that
the equilibrium for the reaction 2HL-*-IgMs
(7S) is not completely in favour of IgMs.
Quite possibly, intramolecular heterogeneity may arise not
as a result of the mode of assembly of the IgM molecule
but rather by the conformation that must be assumed by one
halfmer as it associates with another halfmer for which
it possesses strong noncovalent attractions, Likewise,
those molecules lacking strong noncovalent attractions can
readily form two functional binding sites per 2H-2L chain
subunit. Future experimentation in this area could provide
some rewarding information about intramolecular hetero
geneity.
The most exciting observation concerning the possibility
of conformational differences between opposing binding sites
of the same 7S subunit was seen when tryptic fragments were
obtained from both IgM antibodies, As demonstrated in
Figure 13 only 50% of the expected yield of Fabp's was
obtained by gel filtration on G-200 Sephadex. Approx-


83
imately 90% of these Fahy's absorbed to and were subset
quently hapten eluted from a DNP-lys-sepharose affinity
column. Each of these Fab's contained one site as
measured by equilibrium dialysis (Figure 15) and accounted
for ^ 90% of the binding sites present in the untrypsinized
starting material. The obvious conclusion from these
results is that the Fab's which were digested and therefore
not recoverable were exclusively of the type that contained
no active sites for binding the DNP ligand, This finding
is best explained by the presence of two populations of
conformationa1ly distinct Fab's, It is hoped through future
experimentation with trypsin, or one of the other available
proteolytic enzymes, that conditions which will allow for
near 100% recovery of the Fab's can be ascertained.
Accomplishment of such a task will allow for careful invest
igation at both the structural and functional levels of each
population of Fab's.
The finding of trypsin-sens i tive Fab's might have some
connection with an observation made while attempting to
separate the two putative (good and bad) halfmers. The
50% fraction which passed through, the affinity column after
reduction of the IgM in dilute solution was found to be
extremely labile. Immediate analysis of this fraction
after affinity chromatography demonstrated the fraction was
predominantly (>90%) halfmers (Figure 11). However,
storage at 4C overnight or freezing and thawing of this
fraction produced a protein solution of which only a small


84
percentage was able to be concentrated using negative
pressure dialysis. The remainder of material passed
through the dialysis bag (MW exclusion of 12,000-15,000),
Similar treatment of the 50% hapten eluted fraction had
little effect. It is conceivable that this labile halfmer
fraction, like the trypsin-sensitive Fab's, had obtained
a conformation which when purified in dilute solution was
extremely sensitive to proteolysis.
Recent studies have shown that the structure of the
carbohydrate on the H chain of certain antibodies may
influence the strength of the interaction of those anti
bodies with polymeric antigen (54). Studies suggesting
the importance of the protein structure in determining
glycosylation have also been reported (55,56). These
studies in conjunction with one report of asymmetrical
attachment of carbohydrate to rabbit IgG prompted the
experiments in this study with the antibiotic tunicamycin.
One possible explanation of two distinct conformations for
a single protein could be the presence or absence of carbo
hydrate. Unfortunately, as predicted by previous studies
with Tm (57), secretion of IgM from these cell lines was
dramatically decreased ('v 99%) in the presence of Tm.
Likewise under these experimental conditions no intra-
cellular IgM from Tm-treated cells could be isolated,
Perhaps future endeavors with this antibiotic may determine
a suitable concentration and incubation period which would


85
allow for secretion of unglycosylated IgM. One possible
alternative to this approach might be mechanical stripping
of purified IgM.


CHAPTER IV
SUMMARY AND CONCLUDING REMARKS
The objective of this research was to clearly resolve
the question of intramolecular heterogeneity of ligand
binding by homogeneous IgM antibodies. This has been an
area of controversy for over a decade with relatively few
advancements being made in the last five years. By
utilizing hyhridoma-derived IgM antibodies many of the
problems associated with this unresolved question have
been alieviated. This work also attempted to determine if
intramolecular ligand binding differences are due to
primary structural differences or conformational differences.
Chapter II described a somewhat unusual IgM molecule,
14PAF. This 19S IgM antibody exhibited an average of six
homogeneous relatively high affinity binding sites per
molecule for the DNP moiety. The failure to demonstrate
ten binding sites per pentameric molecule was attributable
to the presence of two different light (L) chains in the
secreted molecules. One of the L chains appeared, based
upon limited amino acid sequence studies, to be the L
chains normally found in MOPC-21, the myeloma protein
secreted by the P3 myeloma line. The other L chains,
presumably derived from the murine spleen cell used in the
fusion, were essential for anti-DNP combining sites.
Analysis of the reductive subunits of the secreted IgM
-86-


87
anti-DNP molecules indicated the presence of two types.
One type (designated active) absorbed to DNP affinity
matrices, contained an average of two homogeneous ligand
binding sites per subunit and did not contain MOPC-21
L chains. The other type of subunit (designated inactive)
did not contain any DNP binding sites and contained only
MOPC-21 L chains.
Polypeptide chain recombination studies with mildly
reduced and alkylated y and L chains from each type of
reductive subunit indicated that the y chains were function
ally identical. Furthermore these in vitro recombination
studies indicated that the noncovalent assembly of the
2y-2L chain subunits was restricted by the L chains in
such a way that the recombinant subunits were homogeneous
in terms of L chains. Equilibrium dialysis studies with
active homogeneous recombinant subunits indicated the
presence of but one ligand binding site per.2y-2L chain
subunit.
Chapter III described the structural and functional
analysis of two additional murine anti-DNP IgM hybridoma
antibodies, NP3-17 Cl-20 and SP2/0 1-64 Cl-12. Each of
these 19S IgM molecules displayed an average of five high
affinity binding sites for the DNP moiety. Furthermore,
> 95% of the reductive subunits of each of these proteins
absorbed to and were hapten eluted from a DNP-lys-sepharose
affinity column. These subunits, when examined by equili
brium dialysis using DNP-e-aminocaproate, indicate each to


88
contain an average of one high affinity binding site,
Structural analysis of each molecule indicated each to be
homogeneous. The most surprising finding with these
molecules was the ability to recover only 50% of Fab's
following trypsin hydrolysis. Interestingly, the 50%
that were recovered accounted for all of the binding sites
present in the starting 7S material. This finding, as
well as limited success with the separation of inactive
reductive halfmers (H-L) which subsequently became active,
indicate that differences in conformation of the binding
sites may account for the observed binding heterogeneity.
Although the study with 14PAF (Chapter II) did not directly
answer any questions regarding intramolecular heterogeneity,
the results of the chain recombination studies (one ligand
binding site per 2H-2L chain recombinant subunit) closely
mimic the natural situation that occurs with the two mole
cules discussed in Chapter III, i.e. one binding site per
2H-2L chain subunit. These findings strongly implicate
the mechanism of assembly as a possible factor in the
generation of conformational differences that could account
for the intramolecular heterogeneity of ligand binding seen
with certain IgM molecules.
It is hoped that these studies have laid a foundation
from which important questions regarding the structure,
function, and intracellular assembly of IgM antibodies may
be answered, as well as uncovering other potentially reward
ing areas of research. Optimistically, extensions of these


studies may result in a wide variety of new approaches
to probing and understanding B cell activation and
regulation.


Full Text
INTRAMOLECULAR HETEROGENEITY OF IgM ANTIBODIES
BY
ROBERT CLAY GILES
A DISSERTATION PRESENTED TO THE GRADUATE
COUNCIL OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1 982

"Take instruction, and not silver,
And knowledge rather than choicest gold.
For wisdom is better than jewels;
And all desirable things can not compare
with her."
Proverbs 8:10-11 (NAS)
The Bible

ACKNOWLEDGEMENTS
First, thanks to Dr. Bruce Glick for his encouragement
to me that I pursue this undertaking and for his assistance
in allowing it to become possible. I would like to acknow¬
ledge the continued support and encouragement of my'áávi sor
Dr. William Clem. His experience and perspective in solving
scientific problems have been a major contribution to my
learning process. I am appreciative of. both his tutorage
and his friendship. I would also like to extend my appre¬
ciation to Dr. Kenneth Berns as Chairman of the Department
of Immunology and Medical Microbiology and the other mem¬
bers of my committee, Dr. George Gifford, Dr. Bill Holloman,
and Dr. Paul Klein, for their time and advice. I would also
like to thank Dr. David Klapper for serving as an advisor
for this study and for his collaboration.
Thanks go out to my fellow graduate students, Terry
Van Dyke, Jim Rusche, and Tom Rowe, for helping to make my
short stay in Gainesville a memorable one. Special thanks
to Erv Faulmann and Marie Hoo'ver for being my friends
through it all.
Thanks to my father and my mother for always giving
of themselves unselfishly and for their continued support
and encouragement during this endeavor of my life. I
would like to thank my wife and closest friend, Jansen,
i i i

for her love and sacrifice during this time of our lives.
Without her understanding and support this work would not
have been possible. I would like to thank my son Clay
for the added joy he has brought to our lives and for
*
not keeping us up too many nights. I look forward to
growing and learning together as a family.

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS . . . iii
LIST OF TABLES vi
LIST OF FIGURES vii
ABSTRACT ix
CHAPTER I BACKGROUND 1
CHAPTER II INTRASUBUNIT HOMOGENEITY IN HETERO¬
GENEOUS IgM ANTIBODIES TO THE DNP
MOIETY DERIVED FROM A MURINE HYBRIDOMA
CELL LINE
Introduction 11
Materials and Methods 12
Results 14
Discussion 34
CHAPTER III INTRAMOLECULAR HETEROGENEITY OF TWO
IgM ANTIBODIES TO THE DNP MOIETY
DERIVED FROM MURINE HYBRIDOMA CELL
LINES
Introduction 39
Materials and Methods 41
Results 49
Discussion 78
CHAPTER IV SUMMARY AND CONCLUDING REMARKS . 86
REFERENCES 90
BIOGRAPHICAL SKETCH 96
v

LIST OF TABLES
TABLE PAGE
1. Amino Terminal Sequences of Light Chains
from Unfractionated 19S and Active and
Inactive 7S Reductive Subunits from 14PAF . . 26
2. Amino Terminal Sequences of Heavy and Light
Chains of IgM Antibody to DNP from a Hybridoma
Line NP3-17 Cl -20 55
3. Comparison of Hapten Elution Profiles of Mildly
Reduced SP2/0 1-64 C1 -12 IgM Antibody from Tm-
and Non-Tm-treated Culture Supernatants ... 75
4. Immunoprecipitation of Tunicamycin (Tm)-
treated and Non-Tm-treated Culture Supernatants
and Cel 1 Lysates 76

LIST OF FIGURES
FIGURE PAGE
1. Equilibrium dialysis of mouse hybridoma
protein 14PAF with DNP-e-aminocaproate .... 17
2. Equilibrium dialysis of DNP-e-aminocaproate
of 7S subunits from 14PAF 20
3. Step-wise elutions of protein 14PAF from
DNP-lys-sepharose affinity column using
four concentrati00^.of hapten 22
4. Alkaline urea polyacrylamide gel electro¬
phoresis of isolated light chains from
unfractionated (A), inactive (B), and active
(C)7Ssubunits 25
5. Equilibrium dialysis with DNP-e-aminocaproate
of 14PAF recombinants using either heavy chains
from active subunits (AH) or inactive subunits
(IH) and light chains from active subunits
(AL) 30
6. SDS-PAGE of recombinants formed using heavy
chains from either active or inactive subunits
combined with an equimolar amount of light
chains consisting of 60% from active subunits
and 40% from inactive subunits 33
7. Mild reduction profile of 19S IgM from
hybridoma SP2/0 1-64 C1 -12 using increasing
amounts of 2-ME from left to right 44
8. Equilibrium dialysis of 19S IgM from
hybridoma SP2/0 1-64 C1 -12 with DNP-e-amino¬
caproate 52
9. Alkaline urea polyacrylamide gel electro¬
phoresis of isolated light chains from (A)
SP2/0 1-64 Cl-12 and (B) NP3-17 Cl-20 .... 54
10. Equilibrium dialysis using DNP-e-aminocaproate
of NP3-17 Cl-20 "7S" fractions resulting from
mild reduction at dilute protein concentra¬
tions (< 1 mg/ml ) 60
11. 10% polyacrylamide gel electrophoresis (SDS)
of NP3-17 Cl-20 "7S11 62
v i i

FIGURE
PAGE
12. 10% polyacrylamide gel electrophoresis of
timed trypsin digestions of SP2/0 1-64 Cl-12
7S reductive subunits 64
13. Sephadex G-200 elution profile of SP2/0 1-64
Cl-12 Fab's obtained after trypsinization
for five hours 67
14. 10% polyacrylamide gel electrophoresis (SDS)
of unfractionated (A) and active (C) Fab's
obtained from five hour trypsinization of
SP2/0 1-64 Cl-12; (B) contains inactive, non-
Fab material 69
15.Equilibrium dialysis of Fab fragments
obtained from a five hour trypsinization
of SP2/0 1-64 Cl-12 with DNP-e-aminocaproate .
1 6
Autoradiogram of specifically purified
â– ^^S-methi on i ne molecules from supernatants
or cell lysates of Tm- or non-Tm-treated
SP2/0 1-64 Cl-12 cells
71
74
v i i i

Abstract of Dissertation Presented to the Graduate
Council of the University of Florida in Partial
Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
INTRAMOLECULARi HETEROGENEITY OF IgM ANTIBODIES
BY
Robert Clay Giles
May, 1982
Chairman: Dr. L. William Clem
Major Department: Immunology and Medical Microbiology
A hybridoma line (14PAF) secreting 19S IgM antibodies
reactive with the DNP moiety was derived from a fusion of
DNP-primed murine spleen cells with the myeloma line P3-
X63-Ag-8. The failure to demonstrate ten binding sites per
pentameric molecule (six were measured) was attributable to
the presence of two different light (L) chains. Analysis
of the reductive subunits indicated the presence of two
types. One type (designated active) contained an average
of two homogeneous ligand binding sites per subunit and did
not contain L chains normally found in MOPC-21, the myeloma
protein secreted by the P3 myeloma line. The other type of
subunit (designated inactive) did not contain any DNP
binding sites and contained only MOPC-21 L chains. Recom¬
bination studies with y and L chains from each type of
reductive subunit indicated that the noncovalent assembly
of the 2p-2L chain subunits was restricted by the L chains

in such a way that the recombinant subunits were homogen¬
eous in terms of L chains. Equilibrium dialysis studies
with active homogeneous recombinant subunits indicated the
presence of but one ligand binding site per 2 p-2 L chain
subunit.
Two additional murine anti-DNP IgM hybridoma anti¬
bodies were derived using the non-immunoglobulin producing
myeloma lines P3-X63-Ag8.653 and SP2/0-Ag-l4. These IgM
antibodies, NP3-17 Cl-20 and SP2/0 1-64 Cl -12, displayed
an average of five high affinity binding sites for the DNP
moiety. Reductive subunits of each of these proteins
absorbed to and were hapten eluted from an affinity column.
These subunits, when examined by equilibrium dialysis, each
contained an average of one high affinity binding site.
Structural analysis of these molecules indicated each to be
homogeneous. Results obtained from trypsin hydrolysis of
each molecule as well as studies performed with reductive
halfmers (H-L) indicated that differences in conformation
of the binding sites may account for the observed binding
heterogeneity.
The results of the chain recombination studies with
14PAF closely mimic the natural situation that occurs with
NP3-17 Cl-20 and SP2/0 1-64 Cl-12. These findings strongly
implicate the mechanism of assembly as a possible factor in
the generation of conformational differences that could ac¬
count for the intramolecular heterogeneity of ligand binding
seen with certain IgM molecules.
¡X

CHAPTER I
BACKGROUND
The structure and physiochemica1 properties of mammal¬
ian IgM immunoglobulins have been elaborated in considerable
detail largely through the study of homogeneous IgM myeloma
proteins. These studies demonstrate that secreted mammalian
IgM antibodies are pentameric molecules containing ten
heavy-light chain pairs and thus, by analogy with IgG
antibodies, should contain ten equivalent ligand binding
sites per molecule (reviewed 1). Although several studies
employing structurally homogeneous IgM monoclonal proteins
have demonstrated the presence of ten homogeneous, rela¬
tively low affinity binding sites per molecule (2,3),
numerous other studies with conventional IgM antibodies
have indicated considerable heterogeneity of ligand binding
with valences of less than ten. In several instances
valences of less than ten were attributable to steric
factors related to antigen size. For example, in one
study the measured valences of IgM antibodies to dextran
progressively decreased as the size of the poly-glucosan
ligand was increased (4). Similarly in another study a
human IgM myeloma protein which bound human IgG was observed
to have an effective valence of five per intact pentamer
(one per subunit) whereas each of the ten Faby fragments
contained an active binding site (5). In other cases,
-1-

2
haptens which are not likely to impose steric limitations
on the antibody combining sites were employed. In this
latter context it is important to point out that an average
of five high and five low affinity binding sites .has fre¬
quently been observed in a variety of species (6,7,8,9).
One study utilizing an unusual IgM subunit containing only
a single heavy-light chain (10) demonstrated that 50% of
these subunits were retained by an immunoadsorbant column
previously used to isolate this heterogeneous antibody from
serum. Since it was also shown that greater than 90% of
the 7S subunits of this IgM antibody were retained by an
immunoadsorbant, the hypothesis was suggested that hetero¬
geneity must exist within individual 7S subunits. However
due to the fact that the IgM was not homogeneous, critics
explained the data by the presence of different populations
of high and low affinity IgM molecules', half of which lack
sufficient affinity as heavy-light chain pairs to be
retained by an immunoadsorbant.
In terms of resolving this issue there would seem to
be two different, but not necessarily exclusive, possibil¬
ities. The first is that ligand binding heterogeneity is
a consequence of differences between antibodies in a popu¬
lation, i.e., intermolecular differences due presumably to
differences in antibody primary structure. The second
possibility is that ligand binding heterogeneity reflects
differences within individual antibody molecules, i.e.,
intramolecular differences. While the first possibility

3
is likely the case in some circumstances wherein obviously
structurally heterogeneous antibodies are employed, the
frequent finding of an average of half high and half low
(% 11 as high as those with high affinity) affinity sites
suggests consideration of the second possibility. Further¬
more, one attempt at separating the two populations of
binding sites suggested that both were on the same IgM
molecule (11), hence adding credence to the possibility
of intramolecular heterogeneity.
The solution to this question has, to a large part, been
elusive due to difficulties in obtaining sufficient amounts
of structurally homogeneous IgM antibodies. A potential
approach to this question was offered by the observations
that the physicochemical properties of immunoglobulins from
lower vertebrates indicate that such animals may be
restricted to but one immunoglobulin isotype analogous to
IgM (reviewed, 12,13). Furthermore, studies using sharks
indicated that certain antigens, especially A-variant
streptococci, can elicit the production of very large
amounts of relatively homogeneous 19S antibodies (up to
10 mg/ml serum) (14,15). Although it has not been possible
to isolate a suitable hapten for studying affinities and
valences with this antigen (16), the finding in sharks of
reasonably good 19S antibody responses to the capsular
polysaccharide of pneumococcal cells and to the DNP hapten
covalently coupled to streptococcal cells provided sufficient
material for limited studies. The results of these studies
with shark IgM antibodies are summarized below.

4
Shark 19S antibodies to the Type III (S^) pneumococcal
capsular polysaccharide were isolated from immune sera by
affinity chromatography and subjected to equilibrium
dialysis using the hexasaccharide hapten. The results
demonstrated several important points: (1) The IgM anti¬
bodies contained an average of ten combining sites per
molecule, (2) in all cases, the antibodies showed marked
heterogeneity of affinities, (3) the antibodies were all
of low average affinities and (4) there was no increase in
the average affinity of the antibodies isolated from any
single animal for periods up to twelve months after initial
immunization. In order to determine if a single IgM mole¬
cule contained ten equivalent combining si tes, the anti¬
bodies isolated from several animals were fractionated by
liquid isoelectric focusing. Equilibrium dialysis experi¬
ments using focused fractions showed the presence of ten
functionally identical combining sites per 19S molecule.
As a proof of the structural homogeneity of focused
fractions, antibodies were mildly reduced, separated into
H and L chains, recombined to 7S subunits (2H-2L chains),
and tested for combining sites by equilibrium dialysis.
The results indicated that these 7S recombinants of focused
antibody fractions each contained two binding sites iden¬
tical to those of the intact antibody whereas heterogeneous
(unfocused) recombinants or isolated H and L chains failed
to show any binding activity. The conclusion from this
study is that the heterogeneity of ligand binding exhibited

5
by nurse shark 19S antibodies to the capsular polysac¬
charide of the Type III pneumococcus can be attributed
to intermol ecu 1 ar heterogeneity most likely at the primary
structural level (17).
In contrast to the results obtained in sharks with the
pneumococcal antigen, those obtained with antibodies to the
DNP moiety dictate a quite different conclusion (18),
Equilibrium dialysis studies using the hapten DNP-e-amino-
caproate with affinity purified nurse shark 19S antibodies
to DNP demonstrated several important points: (1) The 19S
antibodies exhibited heterogeneity of ligand binding with
an average of five high and five low affinity sites per
molecule, (2) the affinities of the low affinity sites were
approximately 1% of those of the high affinity sites, and
(3) no evidence for increased affinities was seen for up to
twenty-one months of immunization. To study the basis for
the ligand binding heterogeneity of the shark 19S anti¬
bodies to DNP, several different approaches were employed.
The results of studies with subunits and proteolytic frag¬
ments showed that neither steric hindrance nor allosteric
effects could account for the observed heterogeneity, i.e.,
the two forms of binding sites were on separable Faby frag¬
ments. In fact these results strongly suggested that both
types of combining sites were present within individual
reductive subunits. Preparative liquid isoelectric focus¬
ing was used in an attempt to separate structurally homo¬
geneous 19S antibodies. The results from equilibrium

6
dialysis studies indicated that each of 16 different focused
preparations contained an average of five high and five low
affinity sites-. These results seemingly argue strongly
against the possibility that the observed heterogeneity was
due to intermolecular heterogeneity. It seems highly
improbable that each of sixteen different isoelectrically
focused anti-DNP preparations would fortuitously be composed
of equimolar mixtures of 19S molecules, half of which have
ten equivalent ligand binding sites approximately 100 times
higher in affinity than those of the remaining low affinity
population. It would seem more likely that the isoelectric
focusing technique actually separated structurally homo¬
geneous antibodies. This interpretation is supported by the
results of recombination studies. Heavy and light chains
from focused molecules yielded 7S recombinants with high and
low affinity sites, albeit in low yields, indistinguishable
from their putative counterparts on the intact molecule.
Since heterogeneous recombinants resulted in binding sites
of only low affinities, it seems likely the focused prepara¬
tions were structurally homogeneous and hence the observed
ligand binding heterogeneity must be an intramolecular
phenomenon (19).
The presence of binding sites of two different affin¬
ities on a single antibody molecule can be explained by
primary structural and/or conformational differences between
the two types of sites. Although the presence of amino acid
sequence heterogeneity in the heavy and/or light chains of a

7
single IgM molecule seems unlikely, the lack of amino acid
sequence data on the shark IgM antibodies makes it impossible
to a^ priori rule out this explanation, Despite this
unclarified point, evidence from another experimental
approach seems to favor the latter possible explanation
mentioned above. Shark 19S antibodies to the DNP moiety
(exhibiting five high and five low affinity sites) and to
the polysaccharide (exhibiting ten low affinity sites)
were each treated with 5 M guanidine-HCl and studied by
equilibrium dialysis with the appropriate hapten after
removal of the denaturing solvent. The results showed this
treatment had no effect on the number or affinity of sites
on the antibodies to the polysaccharide but had a consid¬
erable effect on the antibodies to DNP. In fact the data
obtained with these latter antibodies indicated linear
Scatchard plots that readily extrapolated to a valence of
ten low affinity sites. Thus it appears that the guanidine-
HC1 treatment converted the five high affinity sites to low
affinity ones. This was presumably accomplished by causing
conformational changes in or near the high affinity sites,
Unfortunately sufficient amounts of shark antibodies were
not available to perform detailed studies of the conforma¬
tional relationships between Faby fragments containing high
or low affinity sites.
In light of the suggestion that the intramolecular
heterogeneity of ligand binding by some shark (and perhaps
other species) IgM antibodies to the DNP moiety may result

8
from intramolecular conformational differences, it seems
appropriate to speculate on possible mechanisms responsible
for these putative differences. One possible explanation
is that IgM molecules may be assembled intracel1ularly by
two different mechanisms depending upon the cell involved.
Since the data discussed above suggest these differences
are in fact intrasubunit differences, it is conceptually
**»**•
sound to' suggest that those lymphocytes secreting IgM
antibodies with ten homogeneous sites may do so by assembling
the subunits (2H-2L) from halfmers (H-L) as is the case for
a relatively limited number of IgM myeloma proteins studied
(20,21). In this case it would be predicted that the sub¬
units should exhibit symmetry of ligand binding. On the
other hand, those cells secreting IgM antibodies with half
high and half low affinity sites may do so by assembling
the subunits in a different manner, i.e., either H-H-*H-H-L->
L-H-H-L or H-L+H-H-L+L-H-H-L. If these latter modes of
assembly exist, as is the case with certain other immuno¬
globulin iso types (22), it is conceivable (although admit¬
tedly antidogma) that the formation of the first H-L chain
pair may somehow influence the conformation of the second
pair so as to result in a different affinity site. A priori,
one cannot tell which (high or low affinity) site would be
formed first.
Since the suggestion of intramolecular conformational
differences may seem to some to reflect too much "antidogma,"
it would be appropriate here to briefly consider this issue.

9
There are several reports in the literature which document
the existence of small but detectable conformational dif¬
ferences between Fab fragments derived from specific anti¬
bodies and immunoglobulins (23,24). Admittedly these
proteins were heterogeneous (intermolecular) in primary
structure and hence the existence of such conformational
differences may not be surprising, Furthermore, while the
relatively recent surge of X-ray crystallographic data has
indicated considerable 3-dimensional similarities between
several homogeneous Fab fragments, there are also indica¬
tions of slight differences (reviewed 25), Perhaps the most
important observations that could be cited in support of the
possible existence of intramolecular conformational hetero¬
geneity are those of Edmundson and colleagues (26) with the
myeloma protein and dimer Bence-Jones protein of patient Meg.
These data indicate rather conclusively that different
conformations of a polypeptide chain (L chains in their
study) can be derived from the same amino acid sequence
depending upon the other chain to which the peptide is
paired. Similar precedents have been reported for the
crystalline structures of dimeric insulin (27) and chymo-
trypsin (28). Thus it does not seem absurd at this point to
suggest that such conformational differences could exist and
be functionally important within structurally homogeneous
IgM molecules. Finally, since part of our rationale for
suggesting the role of intramolecular conformational differ¬
ences to explain the apparent intramolecular heterogeneity

10
of ligand binding of some IgM antibodies was based upon the
results obtained with guanidine-HCl treated antibodies, the
results of Richards and coworkers with mouse IgA myeloma
protein 460 become pertinent (29), Their data indicated
that this protein, possessing two ligand binding specifi¬
cities, could be rendered unreactive with one ligand (DNP)
by the guanidine-HCl treatment whereas the affinity for the
other (menadione) was unaffected, Presumably this partial
loss in function resulted from an intracombining site
conformational change. It is unknown if such a change
could be detected by the methodology available.

CHAPTER II
INTRASUBUNIT HOMOGENEITY IN HETEROGENEOUS
IgM ANTIBODIES TO THE DNP MOIETY DERIVED
FROM A MURINE HYBRIDOMA CELL LINE
Introduction
In light of the immunologic importance of IgM it
becomes imperative to develop approaches for obtaining
sufficient amounts of structurally homogeneous mammalian
IgM antibodies with specificities for defined ligands in
order to clearly resolve the question of intramolecular
heterogeneity. The approach that seemed most promising and
was undertaken involved cell fusions (hybridomas). Chapter
II describes the initial results obtained with one murine
hybridoma line secreting 19S IgM antibodies reactive with
the DNP moiety. Although the secreted IgM product was a
structurally heterogeneous molecule (due to the presence of
two different light chains) it appeared to be assembled in
a random fashion from two pools of homogeneous subunits.
Furthermore i_n vi tro recombination studies with the
component polypeptide chains revealed the surprising
findings that a) the subunit homogeneity was L chain
directed and b) each recombinant subunit exhibited but
one active site.

Materials and Methods
Hybridomas
The fusion techniques described by Galfre' et al .
(30) and Gerhard et al. (31) were employed with some modi-
O
fications. Briefly 1 x 10 BALB/c spleen cells (mice
injected 3 days previously with 50 yg 2,4-dinitrophenyl-
ficoll ) and 1 x 10^ myeloma cells (P3-X63-Ag8; derived by
Cotton and Milstein) (32) were mixed and centrifuged at
300g for 8 minutes. Cells were resuspended for fusion in
1 ml of a 50% solution (v/v) of polyethylene glycol (PEG-
1000, J.T. Baker Chemical Co.) in Dulbecco's Minimal
Essential Medium (DMEM, Gibco). This suspension was
further diluted at a rate of 6 ml/min with DMEM over a
period of 5 minutes. The cells were washed and resuspended
in 35 ml of the selective medium whic-h contains hypoxan-
thine, aminopterin, and thymidine (33). The cells were
distributed in 100 yl aliquots into 96 well microtiter
plates (Falcon Microtest II) and incubated at 37°C in a
humidified 5l CO^-95% air mixture. After substantial
growth (^ 10-12 days) supernatants were screened for IgM
antibodies to the DNP moiety using a radioimmunoassay (34).
Cultures positive for anti-DNP antibody were cloned by
limiting dilution in soft agarose (35). Individual posi¬
tive clones ('v 5 x 10® cells) were injected into BALB/c
mice primed with pristane to obtain ascitic tumors. One
such clone, designated 14PAF, was selected for the studies
reported here.

13
Immunochemical Procedures
Mouse antibodies to the DNP moiety were purified from
ascitic fluid by affinity chromatography, freed of hapten,
3
and examined by equilibrium dialysis against H-DNP-e-
aminocaproate as described previously (18). For calculating
protein concentrations of the pentameric IgM antibodies, a
molecular weight of 900,000 daltons and an extinction
coefficient (Eoori ) of 11.9 were assumed.
In one experiment 14PAF was subjected to 5.0 M
guanidine-HCl for one hour at room temperature. The
guanidine-HCl was removed by dialysis against Tris buffered
saline (0.15 M NaCl , 0.01 M Tris-HCl, pH 7.4). These
guanidine-HCl treated antibodies were subjected to equili¬
brium dialysis as previously described.
Mildly reduced 7S subunits of protein 14PAF were pre¬
pared by subjecting purified 19S material to reduction with
0.1 M 2-mercaptoethanol in 0.5 M Tris-HCl, pH 8.0, for one
hour at 22°C followed by alkylation with 0.15 iodoacetamide
for one hour on ice. Gel filtration under nondenaturing
conditions (Sephadex G-200 equilibrated with 0.15 M NaCl,
0.01 M Tris-HCl, pH 7.4) indicated that > 95% of such re¬
duced and alkylated 7S material eluted in a volume expected
to contain ^180,000 dalton proteins; analysis under
denaturing conditions without additional reduction indi¬
cated that >90% of the 7S subunits dissociated into equi¬
molar H and L chains. A small percentage of halfmer (H-L)
molecules was also detected. Mildly reduced and alkylated

14
H and L chains and halfmers were obtained by gel filtering
the 7S subunits on Agarose A5M columns equilibrated with
5 M guanidine-HCl containing 0.01 M iodoacetamide.
Recombinant molecules were prepared from mildly reduced
and alkylated H and L chains as described previously (17).
Briefly, the desired amounts of separated H and L chains
were mixed in 5 M guanidine-HCl containing 0.01 M iodoace-
tamide and concentrated by positive pressure dialysis to
^ 5 mg/ml while dialysing against Tris buffer, pH 7.4.
These recombinants were gel filtered under nondenaturing
conditions and >90% of the UV absorbing material eluted in
a volume expected to contain 180,000 dalton proteins;
electrophoresis of these recombinants in SDS polyacryla¬
mide gels indicated the--presence of equimolar H and L
chains (see Figure 5 for example). Alkaline-urea gel
electrophoresis of extensively reduced L chains was per¬
formed by the method of Reisfield and Small (36). SDS-
polyacrylamide gel electrophoresis was performed according
to the method of Laemmli (37). Amino terminal sequence
studies were performed by Edman degradation using an auto¬
mated Beckman sequenator. PTH derivatives were identified
by high pressure liquid chromatography (38).
Results
The hybridoma line (14PAF) used here readily prolif¬
erated as ascitic tumors in pristane-primed BALB/c mice and
yields of 10-20 ml ascitic fluid were obtained from indivi¬
dual mice. Affinity chromatography of these ascitic fluids

on TNP-lys-sepharose yielded from 5-10 mg/ml antibody.
These isolated antibodies were considered to be exclusively
19S IgM(K) based upon sedimentation velocity measurements,
immunodiffusion analysis with commercial antisera and
SDS-gel electrophoresis studies (data not shown).
Equilibrium dialysis of the isolated 19S antibody
against the hapten 2,4-dinitrophenyl-e-aminocaproate
yielded the Scatchard plot depicted in Figure 1. These
data indicate the hybridoma derived IgM antibody contained
an average of about six binding sites per molecule with an
affinity of 2 x 10^M-^. Furthermore, Sips analysis indi¬
cated a heterogeneity index of 0.98 which presumably
reflected a high degree of homogeneity in the binding
constants of the sites being detected. Equilibrium dialysis
of the 19S antibody treated with 5.0 M Gn-HCl demonstrated
an identical Scatchard plot to the untreated antibody
(see Figure 1).
In order to determine if the observation of about six
binding sites per molecule was due to some peculiar steric
effects, reductive 7S subunits of protein 14PAF were prepared
and studied by equilibrium dialysis. As can be seen in
Figure 2, these 7S subunits appeared to exhibit a ligand
binding pattern identical to that observed with the parent
pentameric molecule, i.e., M . 2 sites with an affinity of
% 2 x 10^M~^ per every two H-L chain pairs. These reductive
subunits were then subjected to affinity chromatography on
DNP-1ysine-sepharose. Approximately 55-60% of the material,

Figure 1 .
Equilibrium dialysis of mouse hybridoma protein
14 PAF with DNP-e-aminocaproate.

o

18
designated as active, absorbed and was hapten-eluted from
the affinity matrix; the other 40-45%, designated as
inactive, did not absorb. Each of these populations of
subunits was examined by equilibrium dialysis. As pre¬
sented in Figure 2, the active subun its .exhibited two homo¬
geneous binding sites identical with respect to affinity to
those seen in the unfractionated material. The inactive
subunits contained no demonstrable binding sites for DNP'.'
The finding that both the active and inactive subunits
could be derived from functional IgM molecules prompted the
hypothesis that the secreted pentameric IgM may be assembled
randomly from these two populations of subunits. Thus, as
an indirect test of this hypothesis, an experiment was
undertaken to attempt fractionating the secreted 19S
molecules into subpopulations with differing numbers of
hapten binding sites. Specifically purified protein 14PAF
was reabsorbed to an affinity column and sequentially eluted
with increasing (arbitrary) amounts of DNP-OH. The eluted
components were freed of DNP-OH, quantified for protein
and studied by equilibrium dialysis. The results, depicted
in Figure 3, indicate that the initial pentameric IgM anti¬
body population was composed of subpopulations with differ¬
ent numbers of binding sites: While this experiment was not
"fine tuned" to the point where it is possible to clearly
say that the pentamers were assembled in a random fashion
from the two forms of subunits, the results are certainly
suggestive.

Figure 2. Equilibrium dialysis with DNP-e-aminocaproate
of unfractionated (•—• ), and active (o—o),
and inactive (&—*&) 7S subunits from 14PAF.

r/c x 10'6
no
o

Figure 3. Step-wise elutions of protein 14PAF from DNP-lys-
sepharose affinity column using four concentra¬
tions of hapten. 18% of the eluted protein was
recovered using 3 x 10-4m hapten; 39% using
1 x 10“3m; 30% using 3 x 10"3m, and 13% using
1 x 10 ¿M.


23
In light of the above finding of two apparently distinct
subpopulations of subunits, the possibility of intramole¬
cular heterogeneity at the structural level was considered.
The first approach to this question involved subjecting the
mildly reduced and alkylated L chains from each subunit to
SDS polyacrylamide gel electrophoresis without additional
reduction. The banding patterns of these mildly reduced
chains were clearly different in that the L chains from
the active subunits exhibited a somewhat slower mobility
than those from the inactive subunits (see Figure 6 for
example); extensive reduction of each of these chains
resulted in mobilities expected of ^ 22,000 dalton peptides.
The second approach involved subjecting extensively reduced
and alkylated light chains isolated from the unfractionated
subunits and the two fractionated subunit subpopulations
to alkaline-urea gel electrophoresis. The gel patterns of
the L chains from the two subpopulations were clearly
distinct (Figure 4). These suggestions of L chain differ¬
ences between the two forms of subunits prompted limited
amino acid sequence studies. The results of these studies
(Table 1) indicated several important points. Firstly, the
L chains from the unfractionated parent molecules exhibited
considerable primary structural heterogeneity with two
different amino acids being demonstrable at 5 (and possibly
7) different positions in the first 12 residues. Secondly,
the L chains from the active and inactive reductive subunits
were each homogeneous (through 12 residues) but quite

Figure 4. Alkaline urea polyacrylamide gel electrophoresis
of isolated light chains from unfractionated
(A), inactive (B), and active (C) 7 S subunits.

25
ABC

26
TABLE 1
Amino Terminal Sequences of Light Chains from
Unfractionated 19S and Active and Inactive
7S Reductive Subunits from 14 PAF
7S subunits
Position
19S unfractionated
Active
Inactive
MOPC 21
1
Asp - Asn
Asp
As n
Asn
2
Val - He
Va 1
He
He
3
Val
Val
Val
Val
4
Met
Met
Met
Met
5
Thr
Thr
Thr
Thr
6
G1 n
G1 n
G1 n
Gin
7
Thr*
Thr*
Ser
Ser
8
Thr - Pro
Thr
Pro
Pro
9
Leu
Leu
Lys
Lys
10
Ser
Ser
Ser
Ser
1 1
Leu - Met
Leu
Met
Met
1 2
Thr*
Thr*
Ser
Ser
The amino
terminal sequence of
the light
chains from
M0PC-21
(Svasti and Milstein, 1971) is shown for comparison.
*Ser could be masked

27
different from each other, A comparison of the amino termi¬
nal sequences of the L chains from these two subpopulations
of subunits with the L chains from MOPC-21 (the L chain
secreted by the myeloma cell line used in the original
fusion) indicated that those from the inactive subunits were
identical (through 12 residues) to those from MOPC-21, The
L chains from the active subunits were quite different.
Hence it seems quite likely that the latter L chains were
encoded for by the genome of the plasma cell used in the
fusion. Therefore the secreted pentameric IgM product of
the hybrid cell contained both MOPC-21 and "anti-DNP" L
chains.
The u chains from both the active and inactive subunits
from protein 14PAF were blocked at the amino terminus
(presumably due to a pyrollidone carboxylic acid) and thus
were not amenable to automated sequence analysis. In an
attempt to ascertain if these p chains were functionally
identical, recombinant non-covalent subunits were produced
between all possible equimolar combinations of separated
mildly reduced and alkylated p and L chains derived from both
types of subunits. Each of these recombinants was gqT
filtered and the 'v 1 80,000 dalton material (representing
>90% of the total) subjected to affinity chromatography
on DNP-lys-sepharose. Greater than 95% of the recombinants
formed with L chains from active subunits and p chains from
either active or inactive subunits were observed to absorb
to (and to subsequently hapten elute from) the affinity

28
columns; less than 5% of either of the recombinants formed
with L chains from inactive subunits absorbed to the columns.
Equilibrium dialysis with these latter inactive recombinants
indicated no detectable combining sites for the DNP group;
similar studies had indicated that the isolated y or L
chains bound no detectable hapten (data not shown). On the
other hand, the active recombinants recovered by hapten
elution from immunoadsorbant columns were each observed,
in duplicate experiments, to have an average of about one
combining site with an affinity identical to that of the
parent molecule (Figure 5). Similarly a single study was
performed utilizing halfmers (covalently linked H-L's) from
active subunits obtained from the leading edge of the H
chain peak on the 5 M guanidine Agarose A5M column. After
removal of the guanidine by dialysis against Tris buffer,
these subunits ('v 180,000 dalton material by gel filtration)
displayed an average of one combining site (identical to
Figure 5). To further prove the functional identity of the
y chains present in the original 19S molecules, mildly
reduced and alkylated y chains from either the active or
inactive reductive subunits were recombined with an equimolar
amount of mildly reduced and alkylated L chains derived from
the original molecule (i.e., %60% "anti-DNP" and ^ 40%
MOPC-21 L chains). Each of these recombinants was then
gel filtered on Sephadex G-200 and the 2H-2L chain recombi¬
nants (representing >90% of the material) were subjected to
affinity chromatography on DNP-lys-sepharose columns;

Figure 5.
Equilibrium dialysis with DNP-e-aminocaproate of
14PAF recombinants using either heavy chains from
active subunits (AH) or inactive subunits (IH)
and light chains from active subunits (AL).
Recombinants made using either heavy chain with
light chains from inactive subunits showed no
detectable binding.

30

31
approximately 40% of the applied material in each case
failed to absorb to the column whereas the remainder ab¬
sorbed and was recovered by hapten elution. The findings
from this experiment were a) the recombinant subunits which
absorbed to the affinity column contained an average of one
binding site identical in affinity to that seen in the
previous active recombinants (Figure 5) and the original
molecules, and b) the active recombinants contained only
L chains with an SDS polyacrylamide gel electrophoretic
mobility characteristic of the "anti-DNP" L chains whereas
the inactive recombinants appeared to contain only M0PC-21
L chains (Figure 6). Soft laser scans of these stained gels
clearly indicated each of the recombinants to be composed
of equimolar u and L chains. In a further attempt to
ascertain if 14PAF p chains preferentially form recombi¬
nants with either of the two forms of L chains, mildly
reduced and alkylated chains from active reductive subunits
were mixed with a two-fold molar excess of 14PAF L chains
(consisting of a 60:40 ratio of "anti-DNP" and MOPC-21 L
chains). The resultant mixture was, without subsequent gel
filtration, then subjected to affinity chromatography on a
DNP-lys-sepharose column; approximately 52% of the material
failed to absorb to the column whereas the remaining 48%
absorbed and was recovered by hapten elution. Again, as in
the above case where equimolar p and L chains were mixed,
the active recombinants contained equimolar p chains and
"anti-DNP" L chains when examined by SDS gel electrophoresis.

Figure 6. SDS-PAGE of recombinants formed using heavy
chains from either active or inactive subunits
combined with an equimolar amount of light
chains consisting of 60% from active subunits
and 40% from inactive subunits. Lane A repre¬
sents unfractionated recombinants; Lane B repre¬
sents recombinants which absorbed to and were
subsequently hapten eluted from a DNP-lys-
sepharose affinity matrix; and Lane C represents
recombinants which did not absorb to the affinity
column.

33

34
The inactive fraction from this experiment contained y
chains and a greater than three-fold molar excess of L
chains; these L chains were present in a 55:45 ratio of
MOPC-21 to "anti-DNP" L chains. The implications of these
results are considered below.
Discussion
The original purpose of the work undertaken here was
to utilize murine hybridomas as sources of homogeneous IgM
antibodies to test the hypothesis that certain IgM anti¬
bodies can exhibit intramolecular heterogeneity of ligand
binding that is not attributable to primary structural
differences. Although the presence of two different L
chains in protein 14PAF precludes its utility in this regard,
the studies reported here did reveal several novel and
potentially important aspects of IgM structure and function.
It was not surprising, based upon the observation of "mixed"
molecules secreted by other hybridoma cell lines derived
from Ig secreting myeloma cells (39), that protein 14PAF
contained two different L chains. It was surprising, how¬
ever, that protein 14PAF was seemingly randomly assembled
from 2y -2L chain subunits that were homogeneous in terms of
L chains. This finding suggests that each type of subunit
may be assembled from y-L chain (or perhaps y-y-L chain)
(40) intermediates which- can only associate with homologous
y-L (or L) chain intermediates to form 2y-2L chain subunits.
The possibility that this putative L chain restriction at
this level is due to the presence of two different y chains

35
is seemingly ruled out by the results of the in vitro
recombination studies. The finding that y chains from
either type of reductive subunit (active or inactive)
could combine with "anti-DNP" L chains to form active
recombinant subunits certainly indicates a high level of
functional similarity in these chains. Similarly, the
observation of homogeneous 2y-2L chain recombinants formed
with a mixture of L chains and y chains from either type
of subunit argues strongly that the intrasubunit L chain
restriction is attributable to the L chains themselves.
In this regard the results obtained from the recombination
experiment using a two-fold molar excess of L chains to y
chains are particularly important in that they show rather
conclusively that either type of L chain can combine equally
well with the y chains. For example if the y chains had a
preference for recombining with the "anti-DNP" L chains,
no y chains would be expected in the inactive recombinant
fraction as they would all be found in the active fraction.
Likewise, if the y chains had shown a preference for the
MOPC-21 chains, >_ 85% of the recombination mixture should
have been found in the inactive recombinant fraction. As
pointed out in the results, 52% of the recombinant mixture
was found in the inactive fraction (hypothetically, one
would predict 53% if assembly was random for each type L
chain). Furthermore, the presence of "anti-DNP" L chains
along with y chains in the inactive mixture demonstrates
that the "anti-DNP" L chains were not a limiting factor in

36
forming recombinants but that the y chains must have an
equal capacity to form inactive recombinants with MOPC-21
L chains or active recombinants with "anti-DNP" L chains,
In addition the presence of ^ 55% MOPC-21 L chains to ^ 45%
"anti-DNP" L chains in the inactive fraction is almost
precisely what would have been expected if assembly of the
recombinants was random for both L chains (hypothetically,
one would predict 57% MOPC-21 L chains and 43% "anti-DNP"
L chains). It thus appears that assembly of 14PAF subunits
i n vitro mimics assembly of subunits i_n vivo both with
respect to ratios of active to inactive subunits and the
homogeneity in terms of L chains. Hence, the conclusion
from this study is that the combination of one of the L
chains with the y chain produced by this cell line results
in an assembly intermediate that will only associate with
a homologous y-L chain pair to form a subunit. Such
restriction may result from different L chain imposed y
chain conformational differences; future work is required
to determine if such putative y chain conformational differ¬
ences are caused by the seemingly large conformational
differences (as manifested by the differences in SDS gel
electrophoretic mobilities) between mildly reduced and
alkylated MOPC-21 and protein 14PAF "anti-DNP" L chains.
A final point of interest, and potential importance,
from these initial studies with protein 14PAF was the
failure to achieve the anticipated number of combining
sites in the active recombinants. The 2y-2L chain in vitro

recombinants formed with y and L chains (or halfmers)
from active 2y-2L chain reductive subunits should have
exhibited two ligand binding sites if the i_n vitro recom¬
bination process worked perfectly, i.e., such as seemingly
was the case during intracellular assembly (see Figure 2).
On the other hand, due to the technical intricacies of such
experimental manipulations, a loss of (or failure to
recover) some of the active sites'Pool'd not be unexpected.
Thus, for the sake of discussion, if 50% of the sites were
lost (as suggested by the data in Figure 5) it would be
expected that such losses might be random and hence about
25% of the recombinants should have two sites, 50% have one
site and 25% have no active sites. In such a case, only
75% of the 2y-2L chain recombinants would be expected to
absorb to an affinity matrix; these absorbed and hapten
eluted recombinants should exhibit an average of 1.3 sites
per molecule. The results obtained with 2y-2L chain recom¬
binants formed with protein 14PAF "anti-DNP" L chains and
either of the two y chains (from active or inactive reduc¬
tive subunits) clearly showed that >95% of the recombinants
absorbed to a DNP affinity column and therefore had at least
one active site. Furthermore equilibrium dialysis with
these active recombinants indicated an average valence of
only one. Hence the conclusion from these results must be
that each 2y-2L chain recombinant had but one active site
for ligand binding. The reason for this rather striking
result is unknown but it would seem appropriate to speculate

38
that it may be attributable to intrasubunit conformational
differences arising during the i_n vitro assembly; perhaps
one mildly reduced and alkylated y-L chain somehow
influenced the conformation of the other pair to result in
a 2y-2L chain recombinant with but one active site for the
DNP moiety. Such conformational differences, if demon¬
strable in the i_n vitro recombinants of protein 14PAF,
would certainly justify future studies regarding this
possible explanation for heterogeneity of ligand binding
by other Ig M antibodies.

CHAPTER III
INTRAMOLECULAR HETEROGENEITY OF TWO IgM ANTIBODIES
TO THE DNP MOIETY DERIVED FROM MURINE
HYBRIDOMA CELL LINES
Introduction
As previously discussed the results obtained by
several different laboratories using IgM antibodies from a
wide variety of sources indicated that some,, but not all,
of these antibodies appeared to exhibit intramolecular
heterogeneity of ligand binding. As mentioned, these
earlier studies were limited by the relatively small amounts
and the structural heterogeneity of the IgM molecules avail¬
able. In order to clearly resolve this question of intra¬
molecular heterogeneity it has become imperative to
develop approaches for obtaining sufficient amounts of
structurally homogeneous mammalian IgM antibodies with
specificities for defined ligands. Chapter II describes
the initial attempt at obtaining a homogeneous anti-DNP
IgM antibody using hybridoma technology. The study of the
molecule obtained, designated 14PAF, resulted in some rather
interesting and novel observations which might be pertinent
to the question of intramolecular heterogeneity. However,
it was felt that this molecule would not, in reality,
permit an adequate assessment of the hypothesis regarding
intramolecular heterogeneity due to the presence of two
different L chains in the secreted pentamer.
-39-

40
This present chapter describes additional cell fusions
which were undertaken using two myeloma lines, NP3 (41)
and SP2/0 (42), which do not produce immunoglobulin. Two
of the cell lines obtained from these fusions have been
designated NP3-17 Cl-20 and SP2/0 1-64 C1 -12. Each of
these cell lines grows as ascitic tumors in mice and yields
moderate amounts ('v 1 mg/ml) of 19S IgM antibodies to the
DNP moiety. Although apparently structurally homogeneous
(as manifested by alkaline urea gel patterns of isolated
L chains from both proteins and by limited amino acid
sequence analysis of H and L chains from one, NP3-17
Cl-20), the hapten binding data obtained for each of these
proteins indicate an average of only five high affinity
binding sites for the DNP group per 19S molecule. Greater
than 95% of the reductive 7S subunits absorbed to and were
hapten eluted from a DNP-lys-sepharose affinity column. •
When examined by equilibrium dialysis, each subunit contained
an average of one high affinity binding site. One experi¬
ment aimed at defining the molecular basis of this observed
binding heterogeneity attempted to determine if asymmetrical
carbohydrate attachment to the molecule is in any way
involved. Tunicamycin, an antibiotic that prevents glyco-
sylation of glycoproteins, was used in an effort to isolate
carbohydrate-free’IgM antibodies and to study their binding
properties.
Other studies attempting to define the molecular basis
of the observed binding heterogeneity involved physically

41
separating the two types of sites, Mild reduction of each
of these IgM molecules yielded predominantly halfmers (H-L)
in dissociating buffers. Experiments designed to disrupt
the non-covalent associations between opposing halfmers
of a 7S subunit, as well as trypsin digestion of both 7S
molecules, indicated that conformational differences may
exist between the two binding site regions of an individual
7S subunit. A proposed mechanism for this heterogeneity
will be discussed.
Materials and Methods
Hybridomas
Cell fusions were performed as described in Chapter II
using the myeloma cell lines P3-X63-Ag8.653 (41) and
SP2/0-Ag-14 (42). After substantial growth, culture
supernatants were screened for IgM antibodies to the DNP
moiety using a radioimmunoassay (34) or passive hemagglutin¬
ation with DNP conjugated sheep red blood cells. Cultures
were cloned in soft agarose (35) and individual positive
clones were subsequently injected into BALB/c mice primed
with pristane to obtain ascitic tumors. Two such clones,
designated NP3-17 Cl-20 and SP2/0 1-64 Cl -12, were selected
for the studies reported here.
Immunochemical Procedures
IgM antibodies to the DNP moiety were purified from
ascitic fluid by affinity chromatography, freed of hapten
by Dowex 1X-8 ion exchange chromatography, and examined by
equilibrium dialysis against H-DNP-e-aminocaproate as

42
described previously (18). For calculating protein concen¬
trations of the pentameric IgM antibodies, a molecular
weight of 900,000 daltons and an extinction coefficient
11
(Eoon„m t ) of 11.0 were assumed.
280nm I cm
Initially, reduced 7S subunits of both proteins were
prepared by subjecting purified 19S material (concentra¬
tions ranged from 3-10 mg/ml) to reduction with .01 M
and .005 M 2-mercaptoethanol for IgM antibodies from
NP3-17 Cl -20 and SP2/0 1-64 Cl -12, respectively. These
levels of 2-ME were determined empirically for each molecule
as that level of reducing agent which yielded the greatest
percentage of subunits containing covalently linked H and
L chains or halfmers (covalently linked 2H-2L chain 7S
subunits could not be obtained; Figure 7). These reduc¬
tions were performed in 0.5 M Tris-HCl, pH 8.0 for one
hour at 22°C followed by alkylation with 0.15 M iodoace-
tamide for one hour on ice. Gel filtration under non¬
denaturing conditions (Sephadex G-200 equilibrated with
0.15 M NaCl , .01 M Tris-HCl, pH 7.4) indicated that ^ 95%
of such reduced and alkylated 7S material eluted in a
volume expected to contain 180,000 dalton proteins;
analysis under denaturing conditions without additional
reduction indicated the 7S subunits dissociated predomi¬
nantly into halfmers (H-L molecules). Subsequently it was
found that halfmers could be obtained in non-denaturing
buffers by mild reduction of dilute protein solutions
(.5-1.0 mg/ml) in .5 M Tris-HCl pH 8.5.

Figure 7.
Mild reduction profile of 19S IgM from hybridoma
SP2/0 1-64 Cl-12 using increasing amounts of
2 ME from left to right. Lane 1, ,001 M;
Lane 2, .005 M; Lane 3, .01 M; Lane 4, .05 M;
Lane 5, .1 M. Profiles of IgM from NP3-17 Cl-20
were similar with .01 M 2 ME being optimum for
the production of halfmers (H-L).

Q £ E I

45
Alkaline-urea gel electrophoresis of extensively
reduced L chains was performed as in the preceding chapter
by the method of Reisfield and Small (36). SDS-polyacryla-
mide gel electrophoresis was performed according to the
method of Laemmli (37).
Amino terminal sequence studies were performed by
Edman degradation using an automated Beckman sequenator.
PTH derivatives were identified by high pressure liquid
chromatography (38).
Tryptic hydrolysis of 7S reductive subunits (5-10
mg/ml) was performed for various time periods following
the protocol of Klapper et al. (43). Briefly a 1:100
enzyme (trypsin, 3-X crystallized, Worthington Biochemical
Corp.) to substrate ratio was established and the tempera¬
ture was maintained at 37°C throughout the digestion.
Trypsinization was done in .25 MTris-HCl, pH 8.3, and
.01 M CaC12 was added to the reaction mixture prior to
adding trypsin (44). Tryptic digestions were halted by the
addition of equimolar amounts of soybean trypsin inhibitor
(45). Faby fragments were isolated by gel filtration (G-200
Sephadex equilibrated with 0.15 M NaCl, ,01 M Tris-HCl,
pH 7.4), Active Fab's were then selected using a DNP-
lysine-sepharose immunoadsorbant. Equilibrium dialysis was
performed on these Fab's as previously described in this
text. A molecular weight of 50,000 and an extinction coef¬
ficient of 13.0 were used to calculate Fab concentrations
(44).

46
Attempts were made to dissociate putative active
halfmers by disturbing non-covalent interactions between
opposing y chains. Mildly reduced antibody was allowed
to absorb to a DNP-lys-sepharose affinity column followed
by exposure of the column to chaotropic or ionic dissociating
agents. Various concentrations of the following agents
were used: guanidine HC1, sodium thiocyanate, acetate
buffer (pH 5.0), isopropyl alcohol, NP-40, arid Tris-HCl
(pH 8.5).
Affinity labeling of reductive subunits from SP2/0
1-64 C1 -12 was attempted to irreversibly block the active
anti-DNP site on each 7S monomer without affecting the
inactive site. A two-fold excess of dinitrof1uorobenzene
(DNFB) and dinitrobenzenesulfonate (DNBS) to antibody was
used. Two milliliters of a 7.2 x 10~^ M concentration of
antibody in .1 M borate buffer, pH 8.3 was allowed to react
for two hours at room temperature with either DNFB or DNBS
_5
at concentrations of 1.44 x 10 M. After two hours an
equal volume of .1 M Tris-HCl, pH 8.0, was added to each
tube to stop the labeling reaction. The antibody was
dialyzed against Tris-buffered saline (.15 M NaCl , .01 M
Tris-HCl, pH 7.4) overnight. The treated antibody was
passed over a DNP-lys-sepharose affinity column and any non¬
absorbing antibody was measured spectrophotometrically at
280nm. It was hoped that when such treated subunits were
placed in 2.0 M guanidine-HCl and allowed to "refold" upon

47
dialysis against Tris-buffered saline that 50% of the
original inactive sites would become active.
Attempts were made to obtain an anti-idiotypic anti¬
serum to IgM from SP2/0 1-64 Cl-12. It was hoped that such
antiserum might detect idiotypic differences between mole¬
cules which could bind hapten and those which could not.
Each of three BALB/C mice was initially immunized intra-
% « * ' * * '
peritoneally with 100 yg of 19S antibody from SP/20 1-64
Cl-12 emulsified in complete Freund's adjuvant. Each
mouse received three additional intraperitoneal injections
of protein without adjuvant over the next two months. One
month into the immunization schedule each mouse received
1 x 10^ SP2/0.14 myeloma cells in order to produce an
ascitic tumor. Three days after the end of the last
immunization ascitic fluid was removed and assayed for
anti-idiotypic antibodies.
Tunicamycin Experiment
Cells. Six x 106 SP2/0 1-64 Cl-12 cells were incubated
in 10 ml of methionine-free Dulbecco's MEM (with L-gluta-
mine, penicillin and streptomycin) with 15% fetal calf
serum. The experiments were run in a humidified atmosphere
of 5% C02:95% air at 37°C.
35
Cell labeling. Cells were labeled with S-methionine
(American Searle, Arlington Heights, IL, 900 Ci/mmole) by
the addition of 100 yCi to 10 ml cultures. When tunicamycin
(Tm) (Eli Lilly, Co., Indianapolis, IN) was employed, cells

48
were suspended in the above medium containing 10 yg/ml
Tm for one hour before addition of the label. Cells were
labeled for eight hours before supernatants were removed
for analysis.
Purification of antigen-specific counts. Cells were
removed from supernatants by centrifugation at 400g for
15 minutes. Cell-free supernatants were passed over a
DNP-1ys-sepharose affinity column to purify antigen-
specific radioactive molecules. This purified antibody
was hapten eluted (.1 M DNP-OH) and subjected to Dowex
1X8 ion exchange chromatography to remove the DNP-OH.
It was necessary at this point to spike the radioactive
IgM with unlabeled IgM in order to avoid significant loss
of radioactivity on the Dowex column. Radioactivity was
assessed by adding 10 y 1 of sample to 2 ml of scintillation
cocktail containing 50% (v/v) Triton-X-100.
Gel analysis. Tm-treated and non-treated purified
antibodies were subjected to SDS-DATD-acrylamide gel chroma¬
tography (46). Gels were dried, overlaid with Kodak X-OMat
AR film and stored between intensifying screens for desired
exposure times.
Sequential hapten elutions. Purified Tm- or non-Tm-
treated antibody was mildly reduced with .1 M 2ME for one
hour and alkylated (.15 M iodoacetamide) for one hour on
ice. These reductive subunits were allowed to absorb to a
small DNP-lys-sepharose column and subjected to step-wise
gradient hapten elutions (beginning with 1 X 10 ^ M DNP-
OH) until all radioactivity was eluted.

49
Analysis of nonspecific radioactivity from supernatants.
Radioactivity from Tm- or non-Tm-treated supernatants was
examined for nonspecific IgM by immunoprecipitating super¬
natant fluid which had been freed of specific antibody by
affinity chromatography. Twenty microliters of a rabbit
anti-mouse y chain antiserum was added to .1 ml of anti-DNP
free supernatant and incubated for one hour on ice. This
was followed by addition of .2 ml of a 50% suspension of
Staphylococcus A (Cowan I strain) and incubated for 15
minutes on ice. This mixture was centrifuged for three
minutes at 15,000 rpm. Pellets were resuspended in 1%
SDS-Tris buffered saline and samples were removed for
scintillation counting.
Analysis of cell lysates. Pelleted cells from Tm-
or non-Tm-treated cultures were resuspended in 4 ml of
.1% NP40 in .04 M Tris-buffered saline with .01 M EDTA,
pH 8.3. Cell debris was removed by centrifugation at
500g for twenty minutes. Supernatants were then freed
of DNP specific antibodies by affinity chromatography.
Both purified antibody and antibody-free supernatants were
assayed by the methods described above.
Results
Functional and Structural Characterization of the IgM
Antibodies
The hybridoma lines utilized here (NP3-17 Cl-20 and
SP2/0 1-64 C1 -12) grow as ascitic tumors in BALB/c mice
and yield moderate amounts 1 mg/ml) of 19S IgM antibodies
to the DNP moiety. Equilibrium dialysis studies using the

50
hapten DNP-eaminocaproate indicate each to contain an
average of five high affinity (Ka^lO^M ^) binding sites
(Figure 8). Furthermore, ^ 95% of the reductive 7S
subunits of each of these proteins absorb to and subse¬
quently can be hapten eluted from a DNP-lys-sepharose
affinity column. These subunits when examined by equili¬
brium dialysis again using DNP-e-aminocaproate indicate
each to contain an average of one high affinity binding
site. Structural features of these two proteins indicate
a considerable degree of homogeneity as might be antici¬
pated using non-producing myeloma cell lines as the parent
line for the fusion process. Alkaline-urea gel electro¬
phoresis of the isolated L chains from each protein revealed
banding patterns indicating considerably restricted hetero¬
geneity (Figure 9). In addition, limited amino terminal
sequence analysis of the H and L chains from the NP3-17
Cl-20 protein indicate each to be homogeneous (Table 2).
The H and L chains from SP2/0 1-64 C1 -12 IgM were not
amenable to sequence analysis (presumably due to blocked
amino terminii; pyrollidone carboxylic acid).
In experiments designed to measure the binding ability
of either of these 19S molecules after treatment with 5.0 M
guanidine-HCl for one hour followed by dialysis against
Tris buffer, it was found by equilibrium dialysis that the
high affinity binding sites were totally destroyed; no low
affinity sites could be detected (data not shown).

Figure 8. Equilibrium dialysis of 19S IgM from hybridoma
SP2/0 1-64 Cl-12 with DNP-e-aminocaproate.
Results for NP3-17 Cl-20 were similar.

52

Figure 9. Alkaline urea polyacrylamide gel electrophores
of isolated light chains from (A) SP2/0 1-64
Cl-12 and (B) NP3-17 Cl-20.


55
TABLE 2
Amino Terminal Sequences of Heavy and Light
Chains of IgM Antibody to DNP from a Hybri-
dome Line NP3-17 CL-20
RESIDUE
H CHAIN
L CHAIN
1
Glu
Glu
2
Val
As n
3
G1 n
Val
4
Leu
Leu
5
Gin
Thr
6
Gin
G1 n
7
Ser
Ser
8
Gin
Pro
9
P ro
A1 a
1 0
Glu
He
1 1
Leu
Met
12
Val
Ser

56
Separation of Halfmers (H-L)
Mild reduction of either of these IgM molecules
resulted in the formation of noncovalently associated
halfmers (H-L) in nondissociating buffers. These mole¬
cules gel filtered on G-200 Sephadex as would be expected
of a 7S, 180,000 dalton protein. However on SDS-PAGE
(10% acrylamide) without further reduction these molecules
migrated as ^90,000 dalton proteins, i.e., halfmers. Upon
subsequent extensive reduction in SDS the ^90,000 dalton
material dissociated into equimolar 'WO.OOO dalton H chains
and ^22,000 dalton L chains. Since these halfmers non¬
covalently associated in nondissociating buffers it was felt
that appropriate denaturing or chaotropic agents might be
able to disturb the noncovalent interactions without
destroying the binding site of the "good" halfmers, there¬
fore allowing elution and recovery of the "bad" halfmers.
Using a "grab bag" approach, various concentrations of the
following agents were used: guanidine-HCl , sodium thio¬
cyanate, acetate buffer (pH 5.0), isopropyl alcohol, NP-40,
and Tris-HCl (pH 8.5). Initially it was observed that two
column volumes of 2.0 M guanidine-HCl would elute 50% of
the "7S" antibody from the DNP-lys-sepharose column with
the remaining 50% being eluted with .1 M DNP-0H. After
dialysis against Tris buffer the guanidine-eluted fraction
would subsequently bind to the DNP affinity matrix.
Furthermore both the guanidine-eluted and the DNP-eluted
material exhibited one binding site per 2H-2L chain

57
subunit. The major problem in interpreting this experi¬
ment was the observation that all of the bound antibody
could be eluted if more than two column volumes of 2.0 M
guanidine-HCl was used. Therefore there are two distinctly
different interpretations of the data: 1) It is possible
that the two column volumes of guanidine-HCl were eluting
preferentially the "bad" halfmers from the affinity column
and that any more than two volumes began to denature and
elute "good" halfmers. This liberal interpretation of the
data implies that if "bad" halfmefs were eluted by the
smaller amount of guanidine-HCl, half of them must have
subsequently become "good" haTfmerS when dialyzed into Tris
buffer. Proof of this interpretation would substantiate the
theory of conformational differences between opposing
binding sites of a "7S" molecule. 2) The more conservative
interpretation (and more likely) of the data would be that
the guanidine-HCl elution was not preferential but rather
nonspecifically eluted 50% of the antibody. All but one of
the other dissociating agents tried either eluted all of
the antibody from the column or was not able to elute any
of the antibody. Only recently it was observed that .5 M
Tris-HCl (pH 8.5) could elute 50% of the "7S" antibody
absorbed to the DNP affinity column. The use of .5 M
Tris-HCl was prompted by an observation which warrants some
discussion at this point. Rather serendipitously, it was
observed that mild reduction in .5 Ml Tris-HCl of either of
the two 19S molecules at dilute protein concentrations

58
(<1 mg/ml) yielded subunits of which 50% would bind to a
DNP affinity matrix (active) and 50% would not (inactive)
providing the protein was left undiluted as applied to the
affinity column. If the reduced mixture was first concen¬
trated ^90% of the antibody would bind to the column.
When examined by equilibrium dialysis the unconcentrated
inactive fraction displayed no binding sites, whereas the
active fraction displayed an average of one site per pre¬
sumed 2H-2L chain 7S subunit (Figure 10). Interestingly,
if the inactive fraction was first concentrated to > 1 mg/ml
a small percentage (.3 sites/2H-2L chain subunit) of high
affinity sites could be generated. SDS-acrylamide gel
electrophoresis of each fraction demonstrated that these
molecules were predominantly halfmers (H-L) in dissociating
buffer (Figure 11).
Hapten Binding by Tryptic Fragments
The finding of only one active hapten binding site in
each reductive subunit from the two hybridoma-derived IgM
antibodies prompted studies aimed at separating "good"
(hapten binding) from "bad" (no hapten binding) Fab frag¬
ments. The strategy employed for this purpose involved
attempts at preparing such Fab's by trypsinizing reductive
subunits. Originally each reductive molecule was subjected
to trypsinization for various times and aliquots were
analyzed by SDS-PAGE (Figure 12). It was observed that
although relatively little digestion occurred within the
first two hours, digestion to Fa by 1 s (defined as ^50,000

Figure 10. Equilibrium dialysis using DNP-e-aminocaproate
of NP3-17 Cl-20 "7S" active (• • ) andinactive
(o O) fractions resulting from mild reduction
at dilute protein concentrations (< 1 mg/ml).
The unfractionated starting material was iden¬
tical to the active fraction. Concentration of
the inactive fraction (®—®) resulted in the
generation of some sites.

o
VO
o
cJ
o
c\j
■o—ex

Figure 11. 10% polyacrylamide gel electrophoresis (SDS)
of NP3-17 Cl-20 "7 S". Lane A, unfractionated;
Lane B, active fraction, and Lane C, inactive
fraction. D, E, and F depict the SDS-PAGE
patterns of samples A, B, and C after extensive
reduction .

62

Figure 12. 10% polyacrylamide gel electrophoresis of timed
trypsin digestions of SP2/0 1-64 Cl-12 7S
reductive subunits. Length of exposure to
trypsin is designated in hours above each
sample. Panel A depicts unreduced samples;
Panel B depicts the same samples upon reduction
with .1 M 2 ME in 1% SDS in a boiling H^O bath
for three minutes.

64
H
L

65
dalton material containing L chains and part of the y
chains) was fairly rapid between 3-5 hours; very little
change was seen over the course of the subsequent 19 hours.
In addition, no F(ab ) ^ ' s were obtained since the inter-H
chain disulfides were cleaved by the mild reduction step
used in preparing the subunits. Large scale digestions
were performed for periods longer than three hours (five
hours for SP2/0 1-64 Cl-12 and 18 hours for NP3-17 Cl-20) .
Very little difference, if any, could be detected in their
digestion profiles (G-200 and SDS-PAGE). The results of
these large scale digestions are represented in Figures
13 and 14. Surprisingly, only 30-35% of the total protein
(^50% of the expected yield) was recovered as Fab's when
the digested material was chromatographed on G-200 Sephadex
equilibrated in Tris buffer. The remainder of the material
was seen as small peptides. However, as demonstrated in
Figure 15, the Fab's which were recovered displayed one
high affinity binding site as measured by equilibrium dialy¬
sis. An important finding was that those Fab's containing
one site accounted for ^90% of the binding sites present
in the untrypsinized starting material. Thus the digested
Fab's seemingly were exclusively of the type that contained
no active sites for binding the DNP ligand.
Asymmetry of Carbohydrate
One possible explanation for heterogeneity of binding
within the pentamer and reductive subunits of these molecules
could be the presence of an asymmetrical attachment of

Figure 13. Sephadex G-200 elution profile of SP2/0 1-64
C1 -12 Fab's obtained after trypsinization for
five hours. An eighteen hour trypsinization
of NP3-17 Cl-20 produced a similar profile.

67
7S
(2 ml f factions)

Figure 14.
10% polyacrylamide gel electrophoresis (SOS)
of unfractionated (A), and active (C) Fab's
obtained from five hour trypsinization of
SP2/0 1-64 Cl-12; (B) contains inactive, non-
Fab material. D, E, and F depict the SDS-PAGE
patterns of samples A, B, and C after extensive
reduction. SDS-PAGE patterns of NP3-17 Cl-20
Fab's were indistinguishable.

6 9
ABC D E F

Figure 15. Equilibrium dialysis of Fab fragments obtained
from a five hour trypsinization of SP2/0 1-64
Cl-12 with DNP-e-aminocaproate. Results from
equilibrium dialysis with Fab's from NP3-17
Cl-20 were identical.


72
carbohydrate (see 47). This possibility prompted an exper¬
iment designed to obtain carbohydrate-free IgM antibodies.
Tunicamycin (Tm), an antibiotic that prevents glycosyl ation
of glycoproteins by blocking the formation of M-acetyl-
glucosamine-lipid intermediates (48), was used to block
glycosylation of the IgM produced by SP2/0 1-64 Cl-12. It
35
was found that culture supernatants from S-methionine
t T ' *
labeled cells treated for eight hours with Tm contained only
1% (15,000 cpm) of the antibody level (1.5 x 10^ cpm) found
35
in supernatants from non-Tm treated S-methionine labeled
cells. Furthermore, the IgM that was purified from the
supernatants of Tm-treated cells appeared to be no differ¬
ent from IgM purified from the supernatants of non-Tm
treated cells. This was evident by an autoradiogram of
purified antibody run on an SDS-DATD acrylamide gel (Figure
16) and by sequential hapten elutions of reductive 7S
subunits (Table 3). Radioactive material which was not
specifically purified by an affinity column was shown by
immunoprecipitation to be free of IgM (Table 4). Attempts
were made to isolate carbohydrate-free antibody from cell
lysates of Tm-treated' cells, but unfortunately, none was
detected, It is not known if lower levels of Tm or other
incubation times might be advantageous for the production
of unglycosylated IgM by this cell line.
Anti-idiotypic Antiserum
In terms of assessing the variability of antibody sites
the utilization of anti-idiotypic antibodies has become a

3 5
Figure 16. Autoradiogram of specifically purified S-
methionine molecules from supernatants or
cell lysates of Tm- or non-Tm-treated SP2/0
1-64 Cl-12 cells.
Lane A and H - Purified antibody from non-Tm-
treated cultures;
Lane B and G - Purified antibody from Tm-
treated cultures ;
Lane C and F - Purified antibody from non-Tm-
treated cell lysates;
Lane D and E - Purified antibody from Tm-treated-
cell lysates.
Samples E - H were reduced with . 1 M 2 ME and
boiled in 1% SDS for three minutes.

74

TABLE 3
Comparison of Hapten Elution Profiles of
Mildly Reduced SP2/0 1-64 Cl -12 IgM Antibody
from Tm- and Non-Tm-treated Culture Supernatants
Antibody from Antibody from ?
Non-Tm-Treated Culture Tm-Treated Culture^
Supernatants Supernatants
(DNP-OH)
Hapten Concentration CPM Eluted 0D280 E1uted3 CPM Eluted 0D280 E1uted
(%
of Total)
(% of Total)
5 x 10"5
M
0
0
0
0
1 x 10"4
M
4200
(10.5)
.275
650
(8.0)
.210
5x10
M
7500
08.8)
.267
1000
(12.5)
.270
1 x 10_J
M
1 7000
(43,0)
.328
3600
(45.0)
.342
5 x 10‘J
M
4800
(12.0)
. 264
650
(8.0)
. 1 80
1 x 10‘¿
M
0
0.
0
0
34000
(85,0)
1 .1
5800
(.73,0)
1 .002
I nactive
Fraction
4000
(10.0)
,2
‘ 1 500
(19.0)
.18
Total
38000
(95,0)
1 .3
7300
(94.0)
1.182
^Loaded 1 ml DNP-lys-sepharose affinity column with 40,000 cpm.
2
Loaded 1 ml DNP-lys-sepharose affinity column with 8,000 cpm.
3
.5 ml of non-radioactive SP2/0 1-64 Cl-12 antibody (O.D.28Q = 2.0) was added
to each radioactive antibody preparation prior to reduction for use as an optical
density marker.

TABLE 4
Immunoprecipitation of Tunicamycin (Tm)-treated and
Non-Tm-treated Culture Supernatants and Cell Lysates
Amount of Rabbit % of Radioactivity
Sample anti-mouse u chain (ul) Precipitated
I.Control Antibody^ 20 100B
40 100
100 100
0 (100 ul Normal 2
Rabbit Serum)
II.Culture medium spiked with Control Antibody 100 100
r
III.Inactive fraction from non-Tm-treated cultures 100 .004
IV. Inactive fraction from non-Tm-treated culture supernatants 0 (100 ul Normal .008
Rabbit Serum)
V. Inactive fraction from Tm-treated culture supernatants 100 .004
VI. Inactive fraction from non-Tm-treated cell lysates 100 .005
VII. Inactive fraction from Tm-treated cell lysates 100 .004
35
A - S-labeled affinity purified antibody from non-Tm-treated culture supernatants.
B - Percentages were normalized to 100% to account for a uniform loss of radioactive material throughout
the experiment due to non-specific stickiness.
C - Radioactivity which was not absorbed by the DNP-lys-sepharose affinity column (i.e. antibody to DNP
removed).
O'»

77
powerful tool (49). The purpose of developing such an
antiserum in this study was hopefully to allow detection
of idiotypic differences between "good" and "bad" halfmers.
Unfortunately no anti-idiotypic antibodies could be detected
in the ascitic fluids of these animals by Ouchterlony
analysis against the intact 19S molecule. Perhaps a better
approach might be to immunize guinea pigs and absorb this
antiserum with the necessary mouse proteins until desired
idiotypic specificity is achieved.
Affinity Labeling
Affinity labeling of various anti-DNP antibodies has
been successfully utilized to either completely or partially
block the antigen combining site via covalently coupling
antigen in the site (50). This approach was . a ttertip.ted with
hopes of obtaining "7S" subunits with inactive sites. By
placing these inactive subunits in 2.0 M guanidine-HCl and
subsequent dialysis against Tris-buffered saline the gene¬
ration of active sites might be possible. This result
would argue that inactive sites have the capability of
binding to antigen but when paired with an active site
assume the wrong conformation for antigen binding. Unfor¬
tunately in this experiment conditions were not appropriate
for affinity labeling as no inactive antibody passed through
the affinity column. It is quite possible that the appro¬
priate conditions for affinity labeling could be achieved
and should be pursued in future studies.

78
Discussion
The purpose of the work described in Chapter III was
to utilize murine hybridomas as sources of homogeneous IgM
antibodies to test the hypothesis that certain IgM anti¬
bodies can exhibit intramolecular heterogeneity of ligand
binding that is not attributable to primary structural
differences. The results of polyethylene glycol induced
fusions using non-immunoglobulin producing cell lines
indicated that stable, antigen specific, IgM secreting
cell lines can be obtained. The two IgM antibodies
obtained from the hybridomas displayed an unusually high
affinity ('v lO^M-"*) for the DNP hapten. It is not known
if cells producing lower affinity IgM antibodies (10 -10 M )
were present as a result of the fusion process and were
missed by the screening procedure used or if, for one
reason or another, cells producing such antibodies simply
did not exist. Nevertheless, these antibodies did meet the
necessary criteria for testing the original hypothesis in
that 1) they were specifically induced pentameric antibodies
which displayed binding heterogeneity, i.e., only five DNP
binding sites. This heterogeneity appeared to be of an
intramolecul ar nature as opposed to intermolecular hetero¬
geneity based upon the following premises. It seems rather
unlikely, due to the nature of hybridoma derived antibodies,
that the two hybridoma cell lines derived (NP3-17 C1 -20 and
SP2/0 1-64 C1 -12) from the two different myeloma cell lines
used (NP3 and SP2/0) could both manufacture and secrete

79
in equal amounts two nondistinct populations of IgM anti¬
bodies, one of which had ten binding sites for DNP and one
of which had no binding sites for DNP, Furthermore,
essentially all (> 90%) of the "7S" re.ductiye. subunits
absorbed to (and subsequently hapten eluted from) a DNP-
lys-sepharose column. These subunits when measured by
equilibrium dialysis displayed an average of one site per
7S subunit. These results clearly support the interpreta¬
tion that each 7S subunit must contain only one site, i.e.,
there were none which contained two and likewise there
were none which displayed no DNP-binding sites. 2) These
IgM antibodies also met the criterion of being structurally
homogeneous, at least by the methods employed. Comparison
of alkaline urea gel banding patterns of myeloma protein
L chains with the banding patterns of these hybridoma L
chains demonstrated considerable structural homogeneity
(for example, compare with the banding pattern of MOPC-21
L chain, Figure 4, Chapter II). Likewise limited amino
acid sequence analysis certainly indicated homogeneity
with NP3-17 Cl - 2 0. Undoubtedly, complete amino acid
sequences of both proteins or sequence analysis at least
through the first hypervariable region would be invaluable
in this regard. However, the question becomes more perti¬
nent when putative inactive sites are separated from active
sites. Perhaps at that stage additional sequence data, as
well as peptide mapping, isoelectric focusing, and/or idio¬
typic analysis would be beneficial.

80
The results from mild reduction of both IgM antibodies
indicated that the 19S molecules proceeded directly from
the 19S covalent pentameric form to covalent halfmers (H-L).
For example Figure 7 demonstrates that a concentration of
.001 M 2ME does relatively little to change the covalent
structure of the molecule whereas a concentration of .005
M 2ME has converted all of the heavy molecular weight
molecules predominantly to halfmers. The obvious inter¬
pretation of this finding is that for these molecules the
inter-heavy chain disulfides are as susceptible to reduction
as are the inter-subunit disulfides. This fact could
become important in uncovering a possible mechanism for
intramolecular heterogeneity. According to Askonas and
Parkhouse (51, p. 632) the intracellular assembly pathway
for IgM "mirrors the resistance of interchain disulphide
bonds to reduction." If this is true with these hybridoma-
derived molecules, it would appear that these molecules are
assembled through a H-L chain intermediate based upon their
reduction profiles. If assembly of IgM is involved in the
observed heterogeneity, comparisons of reduction profiles
of these IgM antibodies with reduction profiles of other
IgM antibodies which possess ten homogeneous binding sites
might bring to light an assembly intermediate (for example
H-H-L, see 40) responsible for the phenomenon.
Examination of the results from experiments designed to
separate noncova1ently associated halfmers yielded few clear
interpretations. This was in part due to the fact that the

81
amount of dissociating agent used (two column yolumes of
2.0 M guanidine-HCl) was critical in removing 50% of the
antibody absorbed on the affinity column. Any more than
this amount or subsequent elutions with the same amount
eluted more antibody from the column. As pointed out
previously the most likely interpretation of these results
would be that the guanidine-HCl elution was not preferen¬
tial but rather nonspec ifi cal 1y eluted 50% of the antibody.
Likewise, results obtained when .5 M Tris-HCl was used to
dissociate the halfmers were equally difficult to interpret
due to the problems associated with total recovery of hapten
binding sites. Neither the 50% fraction which was eluted
from the affinity column by .5 M Tris-HCl nor the 50%
fraction which passed through the DNP-immunoabsorbant when
the mild reduction was performed on dilute solutions of
antibody, displayed any active binding sites by equilibrium
dialysis. The hapten eluted fractions (the remaining 50%)
in both cases displayed only one active site per 2H-2L
chain subunit, thus accounting for only one half of the
original sites in the unfractionated subunit population.
Although equilibrium constants were not measured for the
noncovalent association of halfmers used in this study,
one published report of halfmer association using three
other IgM monoclonal antibodies gives an equilibrium constant
of 2.3 X 10 ^ moles halfmers ^ for the reaction one halfmer
two halfmers (52). It is quite possible that equili¬
brium constants of this magnitude or higher may exist for

82
the hybridoma-derived IgM antibodies described in this
study. If so, separation of the putatiye "good and bad
halfmers" using chaotropic or dissociating agents may be
impossible, as these agents may equally disrupt hapten
binding and halfmer association. Unlike the previous
study mentioned on halfmer association Parkhouse (53*, p. 640)
in describing halfmer association with IgM from the mouse
myeloma MOPC 104E states:
Since, therefore, there is no pronounced
tendency for non-covalent interaction between
HL subunits it is no surprise to find that
the equilibrium for the reaction 2HL-*-IgMs
(7S) is not completely in favour of IgMs.
Quite possibly, intramolecular heterogeneity may arise not
as a result of the mode of assembly of the IgM molecule
but rather by the conformation that must be assumed by one
halfmer as it associates with another halfmer for which
it possesses strong noncovalent attractions, Likewise,
those molecules lacking strong noncovalent attractions can
readily form two functional binding sites per 2H-2L chain
subunit. Future experimentation in this area could provide
some rewarding information about intramolecular hetero¬
geneity.
The most exciting observation concerning the possibility
of conformational differences between opposing binding sites
of the same 7S subunit was seen when tryptic fragments were
obtained from both IgM antibodies, As demonstrated in
Figure 13 only 50% of the expected yield of Fabp's was
obtained by gel filtration on G-200 Sephadex. Approx-

83
imately 90% of these Fahy's absorbed to and were subset
quently hapten eluted from a DNP-lys-sepharose affinity
column. Each of these Fab's contained one site as
measured by equilibrium dialysis (Figure 15) and accounted
for ^ 90% of the binding sites present in the untrypsinized
starting material. The obvious conclusion from these
results is that the Fab's which were digested and therefore
not recoverable were exclusively of the type that contained
no active sites for binding the DNP ligand, This finding
is best explained by the presence of two populations of
conformationa1ly distinct Fab's, It is hoped through future
experimentation with trypsin, or one of the other available
proteolytic enzymes, that conditions which will allow for
near 100% recovery of the Fab's can be ascertained.
Accomplishment of such a task will allow for careful invest¬
igation at both the structural and functional levels of each
population of Fab's.
The finding of trypsin-sens i tive Fab's might have some
connection with an observation made while attempting to
separate the two putative (good and bad) halfmers. The
50% fraction which passed through, the affinity column after
reduction of the IgM in dilute solution was found to be
extremely labile. Immediate analysis of this fraction
after affinity chromatography demonstrated the fraction was
predominantly (>90%) halfmers (Figure 11). However,
storage at 4°C overnight or freezing and thawing of this
fraction produced a protein solution of which only a small

84
percentage was able to be concentrated using negative
pressure dialysis. The remainder of material passed
through the dialysis bag (MW exclusion of 12,000-15,000),
Similar treatment of the 50% hapten eluted fraction had
little effect. It is conceivable that this labile halfmer
fraction, like the trypsin-sensitive Fab's, had obtained
a conformation which when purified in dilute solution was
extremely sensitive to proteolysis.
Recent studies have shown that the structure of the
carbohydrate on the H chain of certain antibodies may
influence the strength of the interaction of those anti¬
bodies with polymeric antigen (54). Studies suggesting
the importance of the protein structure in determining
glycosylation have also been reported (55,56). These
studies in conjunction with one report of asymmetrical
attachment of carbohydrate to rabbit IgG prompted the
experiments in this study with the antibiotic tunicamycin.
One possible explanation of two distinct conformations for
a single protein could be the presence or absence of carbo¬
hydrate. Unfortunately, as predicted by previous studies
with Tm (57), secretion of IgM from these cell lines was
dramatically decreased ('v 99%) in the presence of Tm.
Likewise under these experimental conditions no intra¬
cellular IgM from Tm-treated cells could be isolated,
Perhaps future endeavors with this antibiotic may determine
a suitable concentration and incubation period which would

85
allow for secretion of unglycosylated IgM. One possible
alternative to this approach might be mechanical stripping
of purified IgM.

CHAPTER IV
SUMMARY AND CONCLUDING REMARKS
The objective of this research was to clearly resolve
the question of intramolecular heterogeneity of ligand
binding by homogeneous IgM antibodies. This has been an
area of controversy for over a decade with relatively few
advancements being made in the last five years. By
utilizing hyhridoma’-derived IgM antibodies many of the
problems associated with this unresolved question have
been alieviated. This work also attempted to determine if
intramolecular ligand binding differences are due to
primary structural differences or conformational differences.
Chapter II described a somewhat unusual IgM molecule,
14PAF. This 19S IgM antibody exhibited an average of six
homogeneous relatively high affinity binding sites per
molecule for the DNP moiety. The failure to demonstrate
ten binding sites per pentameric molecule was attributable
to the presence of two different light (L) chains in the
secreted molecules. One of the L chains appeared, based
upon limited amino acid sequence studies, to be the L
chains normally found in MOPC-21, the myeloma protein
secreted by the P3 myeloma line. The other L chains,
presumably derived from the murine spleen cell used in the
fusion, were essential for anti-DNP combining sites.
Analysis of the reductive subunits of the secreted IgM
-86-

87
anti-DNP molecules indicated the presence of two types.
One type (designated active) absorbed to DNP affinity
matrices, contained an average of two homogeneous ligand
binding sites per subunit and did not contain MOPC-21
L chains. The other type of subunit (designated inactive)
did not contain any DNP binding sites and contained only
MOPC-21 L chains.
Polypeptide chain recombination studies with mildly
reduced and alkylated y and L chains from each type of
reductive subunit indicated that the y chains were function¬
ally identical. Furthermore these in vitro recombination
studies indicated that the noncovalent assembly of the
2y-2L chain subunits was restricted by the L chains in
such a way that the recombinant subunits were homogeneous
in terms of L chains. Equilibrium dialysis studies with
active homogeneous recombinant subunits indicated the
presence of but one ligand binding site per.2y-2L chain
subunit.
Chapter III described the structural and functional
analysis of two additional murine anti-DNP IgM hybridoma
antibodies, NP3-17 Cl-20 and SP2/0 1-64 Cl-12. Each of
these 19S IgM molecules displayed an average of five high
affinity binding sites for the DNP moiety. Furthermore,
> 95% of the reductive subunits of each of these proteins
absorbed to and were hapten eluted from a DNP-lys-sepharose
affinity column. These subunits, when examined by equili¬
brium dialysis using DNP-e-aminocaproate, indicate each to

88
contain an average of one high affinity binding site,
Structural analysis of each molecule indicated each to be
homogeneous. The most surprising finding with these
molecules was the ability to recover only 50% of Fab's
following trypsin hydrolysis. Interestingly, the 50%
that were recovered accounted for all of the binding sites
present in the starting 7S material. This finding, as
well as limited success with the separation of inactive
reductive halfmers (H-L) which subsequently became active,
indicate that differences in conformation of the binding
sites may account for the observed binding heterogeneity.
Although the study with 14PAF (Chapter II) did not directly
answer any questions regarding intramolecular heterogeneity,
the results of the chain recombination studies (one ligand
binding site per 2H-2L chain recombinant subunit) closely
mimic the natural situation that occurs with the two mole¬
cules discussed in Chapter III, i.e. one binding site per
2H-2L chain subunit. These findings strongly implicate
the mechanism of assembly as a possible factor in the
generation of conformational differences that could account
for the intramolecular heterogeneity of ligand binding seen
with certain IgM molecules.
It is hoped that these studies have laid a foundation
from which important questions regarding the structure,
function, and intracellular assembly of IgM antibodies may
be answered, as well as uncovering other potentially reward¬
ing areas of research. Optimistically, extensions of these

studies may result in a wide variety of new approaches
to probing and understanding B cell activation and
regulation.

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BIOGRAPHICAL SKETCH
The author was born December 15, 1952, in Tupelo,
Mississippi, as the third child of Rex and Gertrude Giles.
Bob graduated from Tupelo High School in 1970, and attended
Mississippi State University from 1970-1974 on scholarship
as an athletic trainer for the Bulldogs. After receiving
a Bachelor of Science degree in general science with a major
in zoology and a minor in chemistry, Bob enrolled in the
master's program in the Department of Microbiology at
Mississippi State to study under Dr. Lewis R. Brown. It
was also at this time that he was married to Jansen Lamberson.
The author received his Master of Science degree from
Mississippi State in 1976, and remained in the Microbiology
Department for approximately one year as a Research Asso¬
ciate. He enrolled at the University of-FI or ida in the
Fall of 1977 in order to obtain his Doctor of Philosophy
degree in immunology. After acceptance into candidacy for
this degree Bob elected to follow his mentor, Dr. L. William
Clem, to the University of Mississippi Medical Center,
Jackson, Mississippi, where Dr. Clem had recently been
appointed as Chairman of the Department of Microbiology.
While in Jackson, Bob and Jansen were blessed by the birth
of their son Robert Clay, Jr. After graduation from the
University of Florida the author plans to do post-doctoral
work in the laboratory of Dr. J. Donald Capra at the Univer¬
sity of Texas Health Science Center at Dallas, Dallas, Texas.
-96-

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
—
L. William Clem, Chairman
Professor of Immunology and
Medical Microbiology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Professor of Immunology and
Medical Microbiology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosoj
George E. Gifford
Professor of Immunology and
Medical Microbiology

I certify that I have read this study and that in roy
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Assistant Professor of
.Iroroúricfogy and Medical
Microbiology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully a d e q u a t e ,J-rr_slTtx>e and quality,
as a dissertation for the degree on Doctor^ef Philosophy.
C úÚii3
Paul A. Klein
Associate Professor of
Pathology
This dissertation was submitted to the Graduate Faculty
of the College of Medicine and to the Graduate Council,
and was accepted as partial fulfillment of the requirements
for the degree of Doctor of Philosophy.
May 1982
Dean, College of Medicine
Dean for¿7Graduate Studies
and Research

UNIVERSITY OF FLORIDA



UNIVERSITY OF FLORIDA



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