The effect of proteolytic enzymes on the physiological absorption of vitamin B₁₂

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Title:
The effect of proteolytic enzymes on the physiological absorption of vitamin B₁₂
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x, 124 leaves : ill. ; 28 cm.
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Francis, Gary Lee, 1949-
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Trypsin   ( lcsh )
Intrinsic factor (Physiology)   ( lcsh )
Vitamin B12   ( lcsh )
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Thesis:
Thesis--University of Florida.
Bibliography:
Includes bibliographical references (leaves 121-123).
Statement of Responsibility:
by Gary Lee Francis.
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Typescript.
General Note:
Vita.

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University of Florida
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Full Text








THE EFFECT OF PROTEOLYTIC ENZYMES ON THE
PHYSIOLOGICAL ABSORPTION
OF VITAMIN B12










By

Gary Lee Francis


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


1975












ACKNOWLEDGMENTS


My gratitude, appreciation, and respect are extended to the three

individuals, Dr. E.G. Sander, Dr. O.M. Rennert and Dr. P.P. Toskes, who

have made this study possible. Their suggestions, tireless listening,

contributions, and encouragement have been without equal.

I can not adequately thank Mr. George Smith, whose companionship

and assistance have supported this entire effort. Likewise, I wish to

thank my parents, who have lent compassion and understanding when it

was most needed.

Specifically, I would like to thank Dr. Leon Ellenbogen, for his

most generous gift of intrinsic factor. Dr. Gilbert Ashwell and Dr.

Michael Roberts are also to be thanked for their stimulating discussions

and suggestions regarding the carbohydrate nature of intrinsic factor.

I would also personally like to thank Mrs. Carole Quadagno for her sac-

rifice and assistance in the preparation of this manuscript.

This research was supported by the National Institutes of Health

research grant number AM 17866-01. Also, this study was supported by

the National Institutes of Health Medical Scientist Training Program

fellowship number 5 F12 GM56043-03.












TABLE OF CONTENTS


Acknowledgements............................................. ii

List of Tables..................................................iv

List of Figures.................................................. vi

List of Appendices....................... ..................... ix

Abstract...... ............................................ xi

Introduction.....................................................1

Materials......................................................12

Methods.......................................................... 13

Results ........ ............................................... 37

Discussion.....................................................86

Conclusion................... ........... ................96

Appendices...................................................... 98

Bibliography................................................... 121

Biographical Sketch............................................124


















iii











LIST OF TABLES


Table 1. Overall Purification Scheme for the Purification

of Intrinsic Factor and Non-Intrinsic Factor..........46

Table 2. Results of the One-Step Purification Scheme for

Intrinsic Factor and Non-Intrinsic Factor............48

Table 3. Results of Polyacrylamide Disc Gel Electrophoresis

of the Vitamin B12 Binding Proteins..................49

Table 4. Specific Activity of the Vitamin B12 Binding Proteins

Before and After Trypsin Treatment...................52

Table 5. Apparent Equilibrium Constants for the Binding of

Vitamin B12 to the Purified Vitamin B12 Binding

Proteins and Their Trypsin Treated Analogs............55

Table 6. Thermodynamic Equilibrium Constants for the Binding

of Vitamin B12 to the Purified Vitamin B12 Binding

Proteins and Their Trypsin Treated Analogs............56

Table 7. Second Order Rate Constants for the Attachment of

Vitamin B12 to the Vitamin B12 Binding Proteins

and Their Trypsin Treated Analogs....................58

Table 8. First Order Rate Constants for the Dissociation of

the Vitamin B12 Complexes with the Vitamin B12

Binding Proteins and Their Trypsin Treated Analogs....59

Table 9. Polyacrylamide Disc Gel Electrophoresis of the

Vitamin B12 Binding Proteins and Their Trypsin

Treated Analogs....... ... ........................61







Table 10.






Table 11.




Table 12.


Table 13.




Table 14.


Sodium Dodecyl Sulfate Polyacrylamide Disc

Gel Electrophoresis of the Vitamin B12

Binding Proteins and Their Trypsin Treated

Analogs...................................... .......62

Molecular Sieve Chromatography of the

Intrinsic Factor Vitamin B12 Complex Using

G-200 Sephadex.............................. .... ..68

The Effect of Neuraminidase Treatment on the

Vitamin B12 Binding Proteins.........................74

The Effect of Neuraminidase Treatment on the

Vitamin B12 Binding Proteins Stabilized by

Bovine Serum Albumin.................................75

Polyacrylamide Disc Gel Electrophoresis of

the Vitamin B12 Complexes of Intrinsic Factor

and Trypsin Treated Intrinsic Factor and Their

Neuraminidase Treated Analogs........................77











LIST OF FIGURES


Figure I


Figure II


Figure III


Figure IV


Figure V


Figure VI

Figure VII


Figure VIII


Figure IX




Figure X


Figure XI


An Outline of the Probable Passage of Vitamin

B12 Through the Alimentary Tract......................11

A Schematic Outline of the Steps Involved in

the Purification of Hog IF and NIF....................17

Chromatography of the Eluate from AG1-X8 Ion

Exchange Chromatography on QAE-A50 Sephadex............38

Chromatography of Crude Hog IF on the First

Vitamin B12-Sepharose Affinity Column .................40

The Second Vitamin B12-Sepharose Affinity

Column ................ .................... ........... 41

Hydroxyl Apatite Chromatography of NIF.................43

The Separation of IF from NIF on the Third

Vitamin B12-Sepharose Affinity Column ................44

One-Step Procedure for the Purification of

IF and NIF........................................ 47

Determination of the Equilibrium Constant by

Filtration Over G-25 Sephadex for the Binding

of Purified Hog IF to Vitamin B12......................53

Kinetics of Attachment of Vitamin B12 to

Purified IF.................. ........ ..............57

Polyacrylamide Disc Gel Electrophoresis of the

Vitamin B12 Complexes of IF and TIF...................64







Figure XII


Figure XIII


Figure XIV




Figure XV






Figure XVI




Figure XVII




Figure XVIII




Figure XIX






Figure XX


Polyacrylamide Disc Gel Electrophoresis of the

Vitamin B12 Complexes of NIF and TNIF.................65

Polyacrylamide Disc Gel Electrophoresis of the

Vitamin B12 Complexes of Fx4 and TFx4................67

Molecular Sieve Chromatography Using G-150

Sephadex of the Vitamin B12 Complexes of

IF and NIF ......................................... 70

Double Reciprocal Plot for Determination of the

Equilibrium Constant for the Binding of the IF-B12

Complex and the TIF-B12 Complex to the Guinea Pig

Ileal Homogenate at 250C............................ 71

Kinetics of Attachment of the IF-B12 Complex and

the TIF-B12 Complex to the Guinea Pig Ileal

Homogenate at 250C...................................72

Polyacrylamide Disc Gel Electrophoresis of the

IF-B12 Complex and Its Neuraminidase Treated

Analog..............................................78

Polyacrylamide Disc Gel Electrophoresis of the

TIF-B12 Complex and Its Neuraminidase Treated

Analog .............................................. 79

Double Reciprocal Plot for the Determination of

the Equilibrium Constant for Binding of the Trypsin

and Neuraminidase Treated IF-B12 Complexes to the

Guinea Pig Ileal Homogenate at 250C. ...............80

Double Reciprocal Plot for the Determination of

the Equilibrium Constant for Binding of the IF-B12

Complex to the Guinea Pig Ileal Homogenate and to

vii











Figure XXI








Figure XXII


the Neuraminidase Treated Homogenate

at 25C .................................. ..........82

Double Reciprocal Plot for Determination of

the Equilibrium Constant for Binding of the

IF-B12, TIF-B12, IFN-B12, and TIFN-B12

Complexes to the Neuraminidase Treated Ileal

Homogenate at 250C...................................83

Effect of Trypsin and Neuraminidase Treatment

of IF on the Transport of Vitamin B12 Across

the Ileal Cell at 37C...............................85


viii












LIST OF APPENDICES


Appendix

Appendix

Appendix

Appendix

Appendix

Appendix


Appendix VII




Appendix VIII




Appendix IX

Appendix X

Appendix XI




Appendix XII


Abbreviations.............................. .... ... ...98

Modified Charcoal Binding Assay of Gottlieb..........99

Antibody Inhibition Assay...........................101

Polyacrylamide Disc Gel Electrophoresis.............103

Schilling Test.................................... 105

Assay of Tryptic Activity Using Benzoyl-DL-

Arginine-p-Nitroanilide HC1..........................106

Determination of the Equilibrium Constant for the

Binding of Vitamin B12 to IF Using the Method of

Hummel and Dreyer (43)..............................107

Determination of the Equilibrium Constant for the

Binding of Vitamin B12 to IF by Equilibrium

Dialysis.......................................... 109

The Kinetics of Attachment of Vitamin B12 to IF......111

Guinea Pig Ileal Homogenate Receptor Binding Assay...113

Equilibrium Constant for Binding of the IF-B12

Complex to the Guinea Pig Ileal Homogenate After

the Method of Steck and Wallach (46).................115

Assay of Neuraminidase Activity Using Bovine

Submaxillary Mucin. .................................118











Appendix XIII


Appendix XIV


Assay of Protease Activity in Neuraminidase

Preparations Using a-Casein as Substrate..........119

Thiobarbituric Acid Assay of Warren for

Free Sialic Acid..................................120







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.


The Effect of Proteolytic Enzymes on the Physiological
Absorption of vitamin B12
by
Gary Lee Francis
June, 1975

Chairman: Dr. Eugene G. Sander
Co-chairman: Dr. Owen M. Rennert
Major Department: Biochemistry

The objective of these studies was to determine the effect of the

proteolytic enzyme, trypsin, on intrinsic factor (IF) and on the absorp-

tion of vitamin B12. To do this, IF from hog stomach was purified 430x

by affinity chromatography over vitamin B12-Sepharose using a gradient of

guanidine HC1. Two other vitamin B12 binding proteins were also isolated

by this procedure.

Enzite (3x crystalline bovine trypsin immobilized on agarose) was

used to trypsinize the three vitamin B12 binding proteins. Several para-

meters of IF activity were investigated, beginning with the overall phys-

iological effectiveness as defined by the standard 24 hr. urinary excre-

tion test in patient J.P., who has pancreatic insufficiency. Pure IF

resulted in a 24 hr. excretion of only 7.3% of the administered dose while

trypsin treated IF (TIF) raised the excretion to 18.4%. The specific ac-

tivities of IF and TIF were found to be the same, as were their sensitivi-

ties to anti-IF antibody. The interaction of both IF and TIF with vita-

min B12 was investigated in terms of the equilibrium constant for binding

(IF: Keq = 1.0 x 1010 M-1 and TIF: Keq = 0.94 x 1010 M-1) and the rate







constant for association (IF: k1 = 0.80 x 108 M-1 sec-1 and TIF: k1 =

0.78 x 108 M-1 sec-1). The equilibrium constants were essentially the

same as were the rate constants. The size and charge characteristics of

IF and TIF were found to be the same as evidenced by similar Rf values

on polyacrylamide disc gel electrophoresis. The aggregation of IF and

TIF, observed on both Sephadex chromatography and polyacrylamide disc
gel electrophoresis, was also the same. The interaction of the IF-B12

and TIF-B12 complexes with the ileal receptor site (guinea pig ileal ho-

mogenate) was likewise investigated by equilibrium studies (IF-B12: Keq =
5.5 x 109 M-1 and TIF-B12: Keq = 5.6 x 109 M-1) and kinetic studies of

the rate constant of association (IF-B12: kI = 1.5 x 106 M-1 sec-1 and

TIF-B12: k1 = 1.7 x 106 M-1 sec-1). Again, the equilibrium constants

were essentially the same as were the rate constants. Since sialic acid
has been postulated to be required for full IF activity, the effect of

neuraminidase (Clostridium perfringens) on both these preparations was
analyzed and found to be the same. Neuraminidase changed the gel mobil-

ities of both IF and TIF, but did not affect vitamin B12 binding, anti-

body sensitivity, or binding to the ileal receptor homogenate. Finally,

the transport of vitamin B12 across the ileal cell was measured in gut

sac preparations. Compared to non-intrinsic factor, both IF and TIF in-

creased the amount of vitamin B12 transported across the ileal cell by

approximately 800%. Neuraminidase treated IF and TIF behaved in the same
fashion.

The effect of trypsinization on the other vitamin B12 binding pro-

teins was also studied. The only change observed was the apparent trans-

formation of one species of non-intrinsic factor (NIF) into another as

seen in polyacrylamide disc gel electrophoresis experiments.







It may be concluded from these studies, that trypsinization of IF

increases its effectiveness in the in vivo physiological absorption

studies. However, it has not been possible to define a significant

change in the IF molecule following trypsin treatment using any of the in

vitro parameters studied here.











INTRODUCTION
Vitamin B12

Vitamib B12 (B12) is an essential nutrient in the mammalian diet,
although several bacterial systems are capable of its biosynthesis. The
major role of B12 as a coenzyme is twofold (1). First, it is involved in
methyl group transfers in both microorganisms and mammals, primarily in
methionine, acetate and methane formation. Secondly, B12 is necessary
for several hydrogen transfer reactions. These reactions are associated
with rearrangement and are often designated by the sequence in (Eqn. 1).
I I I I
-C -C C
a b <---> a b__1)
I I
H X X H

The hydrogen is transferred by way of an ihtermediate formed by homolytic
cleavage of the C Co bond in which the hydrogen equilibrates with the
two methylene protons of the 5' carbon of the deoxyadenosyl residue of
the vitamin B12 coenzyme. This is facilitated by the presence of an elec-
tron withdrawing substituent "X".
Many of the chemical characteristics of vitamin B12 are known and
permit the study of its binding to intrinsic factor and subsequent trans-
port. The ultraviolet, circular dichroism and fluorescence spectra have
been well defined. Similarly, several spin-labeled cobalamin analogs (2)
have recently been synthesized. Raman spectroscopy (3) has also recently
been utilized to determine differences in the degree of planarity in the
corrin system and its interaction with the central cobalt and axial ligands.
1






2
Vitamin B12 is presented to the mammalian digestive system in only

microgram quantities. Therefore, a system must be operative to extract

this vitamin from tissue protein, to capture it at low concentration,

and finally, to absorb the vitamin in physiological amounts. One of the

central elements in this process is the glycoprotein, gastric intrinsic

factor (IF). Although the entire mechanism of B12 absorption is not cur-

rently known, several of the steps are being characterized by extensive

work in this area.

Intrinsic Factor

Intrinsic factor, a glycoprotein of molecular weight approximately

45,200 47,700 Daltons, is secreted by the gastric parietal cell. This

secretion may be stimulated by several pharmacological agents of which

histalog (a histamine-like compound) is most commonly used in the clinical

setting. Furthermore, in patients with pernicious anemia (P.A.), this

glycoprotein is either not found or is present in reduced levels compared

to the normal. Although pharmacological amounts of vitamin B12 may be

absorbed through other mechanisms, the absorption of physiological amounts

requires intrinsic factor.

In the late fifties and early sixties, a great deal of work was done

to isolate and characterize IF. Several laboratories independently made

porcine mucosal extracts which had IF activity as defined in the standard

vitamin B12 urinary excretion test (Schilling's test). Ellenbogen (4,5)

obtained a preparation from minced hog pyloric mucosa by ammonium sulfate

fractionation and DEAE cellulose chromatography which was active in a

test dose of 0.3 mg in the Schilling test. Later, following further

purification with trypsin and chymotrypsin treatment, molecular sieve

chromatography and rechromatography on DEAE Sephadex, he obtained a







preparation homogenous on starch gel electrophoresis. A family of vita-

min B12 binding proteins was isolated having two major substituents: IF

and hog non-intrinsic factor (NIF). Only the IF fraction remained active

in the Schilling test. Sufficient quantities of this material were

isolated to perform physico-chemical analysis including amino acid

analysis. Ellenbogen also studied the complexation of IF with B12. It

was found that the molecular weight of the complex increased with time to

produce species of approximately 100,000 Daltons. These data are consis-

tent with dimer formation after about ten hours. The binding of B12 to

IF was, however, complete by 120 sec. so that the process of dimerization

was significantly slower than that of binding. This would suggest that

these are two distinct events.

The most recent work on IF is, however, that of Allen and Majerus

(6,7,8) and Allen and Mehlman (9,10). They have synthesized column matri-

ces of vitamin B12 derivatives covalently coupled to Sepharose 4B and, by

the use of affinity chromatography, have isolated highly purified vita-

min B12 binding proteins. Some of their findings include data on the

specific activity of IF and NIF as well as the spectral ratios for the

vitamin B12 complexes with these proteins (10). For IF, the specific

activity is 30.3 ig bound/mg protein and the spectral ratio A280 / A301

= 1.62 when B12 is bound. For NIF, the specific activity is 25.1 ig

bound/mg protein and the spectral ratio A280 / A301 = 2.11 when B12 is

bound. They calculated that one B12 binding site is present per IF mole-

cule and the interaction has a binding constant of 1.5 x 1010 M-1. They

also demonstrated a carbohydrate content of 15% and a molecular weight

of 45,200 to 47,700 Daltons as defined by ultracentrifugation. These

binding proteins were essentially homogeneous by polyacrylamide gel







electrophoresis and greater than 99% of the IF preparation was inhibited
by antibody to IF from a patient with P.A.
The kinetics of binding for vitamin B12 to IF were studied by Wag-
staff (11). Using IF derived from human gastric juice, he obtained a
value for the second order rate constant for the forward reaction of

1.56 x 108 L m-sec-1


Interaction of Intrinsic Factor with Vitamin B12

Many laboratories in the past, including Allen's, have looked speci-
fically at the interaction of vitamin B12 with intrinsic factor. Allen
and Mehlman (9) report a spectral shift when B12 is bound by IF such that
the absorption maximum shifts from 361 nm to 362 nm and there is an asso-
ciated decrease in the total absorbance at 361 nm of 32%. More impor-
tantly, Grasbeck (12) and others report a change in the spectral ratio

A361 / A550 from 3.3 for the free vitamin to 4.1 for the bound vitamin.
In 1971, Olesen, Hippe and Haber (13) did an in-depth study of the
binding of B12 and several structural analogs to the intrinsic factor mole-

cule. They reported that:
1) B12 having immunoglobulin G coupled to the 6 position of
the deoxyadenosyl residue still binds IF.
2) B12 having a lactone produced from the acetamide residue on
the B portion of the corrin ring binds poorly to IF.
3) 812 having adenine substituted for the 5,6-dimethylbenzi-
midazole does not bind IF.
4) 812 having poly L-lysine coupled to the phosphate residue
binds poorly to IF.
These data suggest that the binding involves the 5,6-dimethylbenzimidazole

nucleotide, the B portion of the corrin ring, and somewhat less specifi-

cally, the phosphate residue.







Lien, Ellenbogen, Law and Wood (14) have shown that the 5,6-dimethyl-

benzimidazole is displaced during binding to IF by a single histidine

residue. This was suspected by changes in circular dichroic spectra and

photolysis kinetics, and proven by reaction with diethyl-pyrocarbonate

in which all but one histidine residue in the IF-B12 complex undergo car-

bethoxylation.

Immunological research is currently being used to approach an assay

system for both IF and the IF-B12 complex. There are two types of known

antibody: first, the blocking antibody, and secondly, the binding anti-

body. The blocking antibody, as shown by Ardeman and Chanarin (15),

binds to IF before B12 is bound and will prevent the binding of B12

although it will not dissociate the intrinsic factor vitamin B12 complex

(IF-B12). The binding antibody, as shown by Garrido-Pinson (16), will

precipitate with both free IF and the IF-B12 complex. The use of these

two systems thus allows one to measure both free and bound IF.


Vitamin Bl2 Transport

The physiological absorption of B12 is a complicated process. It is

now known that the IF-B12 complex is bound to the micro-villus membrane

of the terminal ileum. The concept of a specific receptor site for the

IF-B12 complex was perhaps first proposed by Glass (17) and was suspected

because of the species'specificity and the ability of the process to be

saturated. Many investigators have examined the binding by ileal tissue

of intrinsic factor, vitamin B12 and the IF-B12 complex. Okuda (18) stud-

ied the binding of IF-B12 to the rat ileum using intestinal sacs isolated

surgically. He found this to be a time dependent process requiring 45

min. for maximal binding to occur. Mackenzie and Donaldson (19), using

isolated micro-villus membranes, showed the binding of IF-B12 to be






6
inhibited at pH values less than 5.4 and by disodium ethylenediamine-

tetraacetate (EDTA) at a concentration known not to cause nonspecific

membrane damage. Furthermore, the EDTA effect was reversible with cal-

cium. This suggested the possibility of calcium bridges or the stabili-

zation of a conformation by calcium thus allowing binding to proceed.

Rothenberg (20) demonstrated that a crude homogenate of the guinea

pig ileum, prepared in Ringer's solution, was capable of binding the

IF-B12 complex. This material was precipitable in 15% sodium sulfate,

inactivated at 560 for 30 min., sensitive to EDTA, unstable at pH less

than 2.0 and excluded by G-200 Sephadex. Most importantly, this binding

protein was isolated only from ileal tissue and not from jejunum, sug-

gesting a physiological role for the ileal uptake. Finkler and Hall (21)

have shown the binding of the IF-B12 complex to the guinea pig ileal horrioo-

enate to be sensitive to disulfide reducing agents and this activity is

partially restored following reoxidation with atmospheric oxygen. Donald-

son et al.(22) have estimated the number of binding sites to be 8.5 x 1011

per hamster ileum. Hooper et al. (23) have shown, using IF isolated by

affinity chromatography and ileal mucosal homogenates, that the binding

of IF-B12 is not inhibited by 100-fold excesses of free B12 or IF. Final-

ly, Katz and Cooper (24) have been able to solubilize a component of the
ileal homogenate using Triton X-100 and sonication which binds IF-B12, is

excluded by G-200 Sephadex and is not inhibited by phospholipase A, neura-

minidase or sulfhydryl blocking agents.

Mathan (25) has examined the kinetics of attachment of intrinsic

factor bound cobamides to the guinea pig ileal receptor. Using cyanoco-

balamin-IF complexes, the second order rate constant for attachment is

1.3 x 106 M-1sec-1. Most interesting, however, is the finding that the




7

binding of other substituted cobamide-IF complexes to the receptor site

is independent of the substitution as long as IF still binds the cobamide.

This would suggest that the region of the IF-B12 complex recognized by

the receptor does not include the geometry of the B12 ligand.

The function of the carbohydrate moiety on IF has long been a subject

of speculation. No definite consensus has been reached and the views have

ranged from no function at all to that of binding to the receptor site.

At least with regard to other glycoproteins, the cellular recognition

apparatus appears to be located in the carbohydrate portion of the mole-

cule. Specifically, the work of Morell and Ashwell (26,27,28,29) with oro-

somucoid and several other serum glycoproteins, has shown that the removal

of sialic acid from these glycoproteins mediates their uptake by hepato-

cytes. The receptor for these glycoproteins can be isolated and sialic

acid must be present on this receptor for it to be functional. In gen-

eral, the binding of these proteins to the receptor site is calcium depen-

dent (3 to 10 nM), pH specific (pH 6 to 8), and sensitive to both dithio-

threitol and neuraminidase.


The Fate of Intrinsic Factor Complexed with Vitamin B12

The exact nature of what happens once IF-B12 has been bound to the

ileal mucosa is still unknown. Several people have investigated this area.

In 1964, Boass and Wilson (30) recovered radiolabeled B12 from homog-

elized ileal tissue and postulated that it was taken up by pinocytosis.

Ukyo and Cooper (31) later showed that intrinsic factor activity could

be recovered from guinea pig ileal homogenates from one to three hours

following administration of IF-B12. The major criticism of this work,

however, is the fact that the IF may have been bound to the micro-villus

membrane. Yamaguchi (32) attempted to answer this question with 51Cr




8
labeled IF and, although he could demonstrate no uptake of the label into

the absorptive cells, he could also not demonstrate that the label had not

been hydrolyzed off during this process. Mackenzie et al. (33) performed

a similar study using 1251 labeled IF and obtained the same results.

Unfortunately, the same criticism is applicable to this study as well.

Thus, whether or not B12 is split from IF still remains in question with

regard to the absorptive cell surface.

Releasing Factor

Several authors have looked for a factor which would release IF from

the IF-B12 complex, presumably once it is bound to the receptor surface.

Herbert and Kaplan (34) claimed to have isolated a factor from intestinal

aspirates which would specifically release IF from the IF-B12 complex.

This was later shown to be the equilibration of free, unlabeled vitamin

B12 with labeled vitamin B12 bound to IF. In 1969, Temperley (35) iso-

lated an inhibitor of IF which he believed was the releasing factor. It

was sensitive to papain, capable of dissociating an IF-B12 complex to

free B12, stable at 1000C for 10 min., destroyed by pH less than 3.5 or

greater than 11.0 and had a molecular weight of approximately 40,000 Dal-

tons. Certainly, the nature of this step in the absorptive pathway for

vitamin B12 remains to be elucidated.

The Role of the Pancreas in Vitamin B12 Absorption

The study of the role of the pancreas in the absorption of vitamin

B12 has been most extensively studied by Toskes et al. (36,37,38) and

Deren et al. (39). Their work has centered around the study of a group

of patients with pancreatic exocrine insufficiency who demonstrate vita-

min B12 malabsorption. In general, they have been able to demonstrate






9
the following findings regarding these patients:

1) All these patients exhibit pancreatic exocrine insufficiency

as evidenced by steatorrhea and an abnormal secretin test

or pancreatic calcification.

2) About 40% of these patients demonstrate vitamin B12 malab-

sorption as defined by less than 8% excretion in the stan-

dard Schilling test.

3) All the patients defined by the first two categories exhibit

no response to hog IF in a dosage which is sufficient to cor-

rect the vitamin B12 malabsorption in a patient with perni-

cious anemia.

4) These patients secrete normal amounts of IF which is func-

tional in a Schilling test.

5) Orally administering trypsin or chymotrypsin to these

patients corrects their B12 malabsorption.

6) Giving the patient his own isolated gastric juice has no

effect.

7) Incubating his own isolated gastric juice with insolubilized

trypsin and chymotrypsin and giving this treated juice to

the patient corrects his absorptive defect.

8) Treatment of hog IF with the same enzymes and administering

this product to the patient corrects his defect.

9) A factor isolated from the pancreas which corrects the mal-

absorption fits the molecular weight and temperature sensi-

tivity of trypsin. Thus, it seems likely that proteolytic

enzymes play a role in the absorption of B12 but exactly

where in the scheme they act is still in question.





10
The overall scheme for the absorption of vitamin B12 may be summar-

ized as seen in Figure I. There are many points along this pathway in

which proteolytic enzymes may play a role. First, vitamin B12 is in-

gested bound to tissue protein and hydrolyzed free in the acid environ-

ment of the stomach. Secondly, IF must bind to free B12 and this inter-

action may be altered by proteolytic activity. Next, the IF-B12 complex

must pass through the alimentary tract where it may aggregate and come

into contact with a multitude of other proteins and factors which may

interact with IF-B12 in an undefined manner. Proteolytic enzymes may

alter these phenomena. In the terminal ileum of man, IF-B12 must bind

to the specific receptor site and this recognition may be altered by pro-

teolytic activity. Either IF-B12 or a component of the original complex

must then be transported, perhaps following interaction with releasing

factor, across the epithelial cell and appear in the portal circulation

bound to trans-cobalamin II. Again, proteolytic enzymes may alter the

ability of IF-B12 to undergo any of these transformations. To begin to

understand the role which they might play, one must first purify IF and

study the effect of proteolytic enzymes on this molecule and its inter-

action at each step in the absorptive pathway.

























? Other Binders
? Inhibitors


Terminal Ileum


IF-B12
Receptor

?B12
^12

TC II TC II-B12



FIGURE I An Outline of the Probable Passage of Vitamin B12 through the

Alimentary Tract












MATERIALS


Hog intrinsic factor (lOx concentrate) was obtained as a generous

gift from Leon Ellenbogen at Lederle Laboratories (Lot #48034-28). This

material was chosen as it is a saline extract from minced pyloric mucosa

and has not been exposed to proteolytic activity. Trypsin (3x crystal-

line) immobilized on carboxy-methyl cellulose (Enzite) was obtained from

the Miles Servac Corporation. Neuraminidase (Type VI, chromatographically

purified) from Clostridium nerfringens was purchased from the Sigma Chem-

ical Company. Neuraminidase immobilized on beaded Agarose was also ob-

tained from the Sigma Chemical Company. Antibody to intrinsic factor was

used as untreated serum from patient K.E. who has pernicious anemia.

Male Hartley guinea pigs weighing less than 250 gis were used in the prep-

aration of the ileal homogenates and gut sacs. [57Co]cyanocobalamin

(B12) was obtained from Amersham Searle Corporation at a specific activity

of either 15 pCi/ig or 100 pCi/pg and, where necessary, was diluted with

crystalline cyanocobalamin from the Sigma Chemical Company. Bovine serum

albumin was Cohn fraction V obtained from the Sigma Chemical Company.

Bovine submaxillary mucin, used to assay neuraminidase activity, was

Type I from Sigma Chemical Company. The a-Casein used in the assay for

protease activity was obtained from the Sigma Chemical Company and was

suitable as a substrate for fibrinolysin. L-Tyrosine also came from the

Sigma Chemical Company. Sepharose 4B was obtained from the Pharmacia

Chemical Corporation.












METHODS

Preparation of the Vitamin B12 Sepharose Affinity Column

Acid hydrolysis. Crystalline vitamin B12 (5.88 gms) along with 1.0 PCi

[57Co]cyanocobalamin was dissolved in 0.4M HC1 to a final volume of 500

ml. This was sealed and stirred for a total of 64 hrs. in the dark.

This entire sample was then stored at -700C until it could be chromato-

graphically purified. The product of this reaction is the vitamin B12

acid hydrolysate.

Chromatography on AG1-X8 ion exchange resin. The vitamin B12 acid hydro-

lysate was chromatographed on Biorad AG1-X8 ion exchange resin. A column

of Biorad AG1-X8 (acetate form) 100 200 mesh ion exchange resin was

poured at 40C to a final column bed volume of 4.5 x 60 cm. This material

was then washed with 2.0 liters 1.OM sodium acetate followed by 3.0 liters

glass distilled water. The vitamin B12 acid hydrolysate was pumped onto

the column at a constant flow rate of 100 ml/hr. After sample applica-

tion, the column was developed by washing with 3.0 liters 0.025M acetic

acid. A peak of radioactivity was eluted in the first 1600 ml. This

material was deeply red in color and is referred to as the vitamin B12

carboxylic acid derivatives. After this material was eluted from the

column, the ion exchange resin remained red tinged. The nature of the

vitamin B12 derivatives remaining bound to the resin was not further in-

vestigated. The solution of vitamin B12 carboxylic acid derivatives was

evaporated to dryness using a rotary evaporator. The temperature was

controlled at 50C. The residue was then dissolved in glass distilled

13





14

water and evaporated to dryness at 50C. This second residue was again

dissolved in 400 ml glass distilled water and, at 600C, was evaporated to

dryness. This final residue was dissolved into 300 ml glass distilled

water. The pH of this solution was 4.7. Pyridine (55 ml) was added and

the pH of this solution was adjusted to 9.0 with drop-by-drop addition of

concentrated ammonium hydroxide (about 1.0 ml). This material was imme-

diately chromatographed using QAE-A50 Sephadex ion exchange resin.

Chromatography on QAE-A50 Sephadex. QAE-A50 Sephadex ion exchange resin

was swollen for 60 hrs. at room temperature in 3.0 liters 1.OM ammonium

acetate. The settled beads were decanted five times using this same buf-

fer. To the settled beads was then added 1.0 liter 0.2M pyridine (pH ad-

justed to 9.0 with concentrated ammonium hydroxide). The beads were

washed five additional times in this buffer and a column (2.5 x 80 cm)

was poured. The column was then equilibrated with 1500 ml 0.2M pyridine

(pH adjusted to 9.0 with concentrated ammonium hydroxide).

After application of the AG1-X8 column eluate the column was washed

with 1500 ml 0.4M pyridine until the elution radioactivity ceased. The

column was developed with a linear 2.0 liter gradient (1.0 liter 0.4M

pyridine 1.0 liter 0.4M pyridine + 0.16M acetic acid) followed by flush-

ing with 1.0 liter 0.40M pyridine + 0.32M acetic acid.

The second peak of radioactivity was pooled and titrated with

1 x 102M sodium hydroxide. This peak was also subjected to high voltage

paper electrophoresis in a 0.05M K2HPO4 buffer, pH 6.50, containing 0.1

gm KCN per liter. Electrophoresis was conducted on Whatman 3MM paper at

a constant voltage of 1000V. This second peak contained the vitamin B12

monocarboxylic acid derivative.

Preparation of 1,6-hexanediamine linked Sepharose 4B. Sepharose 4B (200





15
ml packed volume) was washed five times with water and then mixed with

200 ml 50% dimethyl-formamide (DMF) in water. To this slurry was then

added 50 gms finely crushed CNBr. The pH was adjusted to 11 with the addi-

tion of 8M NaOH and the temperature was kept at 20C with the addition of

ice. After 15 min. when the evolution of hydrogen ions had markedly de-

creased, the slurry was poured into a 600 ml sintered glass funnel, vacuum

filtered to near dryness and immediately washed with 2.0 liters 0.1M

NaHCO3, pH 10.0. At the end of 90 sec. the funnel was clamped off and 0.8

mole 1,6-hexanediamine in 400 ml 50% DMF containing 0.1M NaHCO3, pH 10.0

was added. The slurry was transferred to a flask and stirred at 4C for

24 hrs.

This substituted Sepharose was then washed with 1.0 liter water fol-

lowed by 1.0 liter 0.lM glycine ethyl ester, pH 10.0, and then with 1.0

liter 0.1M glycine ethyl ester, 1.OM NaCl, pH 10.0. The final washing was

accomplished with 4.0 liters'glass distilled water. This material (1.0 ml

packed bed volume) was then titrated with 1 x 102M HC1.

Coupling with vitamin B12-carboxylic acid derivatives. Packed 1,6-hexane-

diamine linked Sepharose (50 ml) was mixed with 500 pmole vitamin B12-

monocarboxylic acid derivative (2.5 x 10-5 pCi/pmole) and 1.0 gm l-ethyl-3-

(3-dimethylaminopropyl)-carbodiimide in 100 ml 50% DMF. The pH was adjust-

ed to 5.6 with NaOH and the slurry was slowly stirred at 40C for 24 hrs.

At this time, another 1.0 gm carbodiimide was added and the pH readjusted

to 5.6. After a total of 64 hrs. of reaction, the slurry was decanted and

the Sepharose washed with the same solutions as in the preparation of 1,6-

hexanediamine linked Sepharose. An aliquot (1.0 ml packed volume) was

removed and the radioactivity contained in it was counted. The entire

Sepharose was deep red in color. This material was thereafter referred







to as vitamin B12-Sepharose.

Purification of Hog IF and NIF

First vitamin B12-Sepharose affinity column. Many steos were utilized in

the purification of IF and NIF. A schematic diagran to summarize these

methods and the fractions involved in each is outlined in Figure II.

Twenty-five gms hog IF (lOx purified by (NH4)2SO4 precipitation) was

suspended into 750 ml O.1M Tris-acetate buffer, pH 9.2, by stirring at

4C for 90 min. This suspension was centrifuged at 20,000 x G for 20 min.,

the supernatant solution decanted away and the pellet resuspended in 0.1M

Tris-acetate, pH 9.2, and recentrifuged. The supernatant solutions were

pooled and vacuum filtered through celite to yield a crude extract of

golden yellow color.

The entire crude extract was pumped onto a 50 ml vitamin B12-Seph-

arose column (1.32 pmole B12/ml) at a flow rate of 15 ml/hr. After sale

application, the column was washed with 0.1M K2HPO4, pH 7.5, until the

absorbance at 280 nm returned to zero (1.0 liter). The column was then

washed with 2.0 liters 0.1M glycine, O.IM glucose, 1.0M NaC1, pH 10.0,

followed by 1.0 liter 0.1M K2HPO4, pH 7.5. The final elution was accom-

plished with 0.1M K2HP04, 7.5M guanidine HC1, DH 7.50 (1.0 liter). The

vitamin B12 binding activity, as assayed by the modified charcoal binding

assay of Gottlieb (40) (Appendix II), was contained in the 7.5M guanidine

HC1 washing. This colorless solution was pooled, dialyzed exhaustively

against glass distilled water and concentrated at 30 PSI using a contin-

uous flow Amicon concentration cell equipped with a PM-10 membrane. The

final volume after concentration was 100 ml.

Second vitamin B12-Sepharose affinity column. The concentrated material

isolated from the first vitamin B12-Sepharose affinity column was imme-












Crude Extract

First Vitamin B12-Sepharose Affinity Column

(B12 binders eluted in 7.5M guanidine HC1)

Second Vitamin B12-Sepharose Affinity Column


(NIF in 7.5M guanidine HC1) (IF in 3-6M guanidine HC1)


Hydroxyl Apatite Hydroxyl Apatite


(NIF in low (Fx4 in high Third Vitamin B12-Sepharose
K2HPO4) K2HPO4)
Sl Affinity Column


(IF in low guanidine HC1)


FIGURE II A Schematic Outline of the Steps Involved in the Purification
of Hog IF and NIF





18

diately reapplied to the same column matrix (1.32 pmole vitamin B12/ml).

This column matrix was washed with 30M ml 7.5M guanidine HC1, 0.1M K2HPO4,

pH 7.5; followed by 500 ml O.M glycine, 0.1M glucose, 1.OM NaC1, pH 10.0;

and lastly, with 1.0 liter 0.l~ K2HP04, pH 7.50, just prior to chromato-

graphy of the vitamin B12 binding proteins to remove any free vitamin B12-

The vitamin B12 binding fraction was pumped onto the column at a

flow rate of 15 ml/hr. followed by successive washes of 500 ml 0.1M K2HPO4,

pH 7.50; then 600 ml 0.1M glycine, 0.lM glucose, 1.OM NaC1, pH 10.0; and

lastly 400 ml 0.1M K2HPO4, pH 7.50. Rather than eluting all the vitamin

B12 binding activity in one step as was done with the 7.5M guanidine HC1,

0.1M K2HP04, pH 7.50 washing of the first affinity column, the vitamin B12

binding proteins were fractionated in this experiment by a series of buf-

fers, each containing higher guanidine HC1 concentrations. Sufficient

quantities of each buffer (4 column volumes or 200 ml) were flushed

through the column to return the 280 nm absorbance of the eluate to about

zero prior to the application of the next buffer. These seven buffers

were composed of 0.lM K2HPO4, pH 7.50, containing respectively, 1.0, 2.0,

3.0, 4.0, 5.0, 6.0 and 7.5M guanidine HC1.

The 7.5M guanidine HC1 step contained exclusively NIF as determined

by the antibody inhibition assay (40)(Appendix III) and was used for fur-

ther purification of NIF. The material eluted in the range from 3.0 to

6.0M guanidine HC1 contained both IF and NIF as determined by the antibody

inhibition assay and was used for the further purification of IF. Both

these fractions were dialyzed exhaustively against glass distilled water

in preparation for the next step.

Hydroxyl apatite chromatography of NIF. The dialyzed fraction from the

second vitamin B12-Sepharose affinity column which was eluted in 7.5M





19
guanidine HC1 was applied by gravity flow to a hydroxyl apatite column

(2.5 x 30 cm) previously prepared in distilled water. After sample appli-

cation, the column was initially eluted with a linear 2.0 liter gradient

(1 liter H20 1 liter 0.05MK2HP04, pH 7.0), followed by a steeper 1.0

liter linear gradient (500 ml 0.05M 500 ml l.OM K2HPO4, pH 7.0). Two

peaks of vitamin B12 binding activity were obtained. The first, which

contained NIF, was rechromatographed on hydroxyl apatite to a constant

specific activity of 9.50 pg vitamin B12 bound/mg protein as measured in

the charcoal binding assay. This protein, when completed with a five-

fold excess of vitamin B12 and passed over a G-25 Sephadex column, had a

specific activity of 17pg vitamin B12 bound/mg protein.

The second peak of vitamin B12 binding activity contained an unre-

ported vitamin B12 binding protein which is hereafter referred to as Fx4.

Both these fractions were dialyzed, concentrated with an Amicon PM-10

ultrafilter, and stored at -70C until used.

Hydroxyl apatite chromatography of IF. The material eluted in the 3.0 to

6.0M guanidine HC1 buffers of the second affinity column was pooled, dia-

lyzed exhaustively against distilled water, and chromatographed on a

hydroxyl apatite column (2.5 x 30 cm) in a fashion identical with that

outlined in hydroxyl apatite chromatography of NIF.

Third vitamin B12-Sepharose affinity column. The third affinity column

was used to remove the last contamination of NIF from the IF preparation.

Vitamin B12-Sepharose was diluted with Sepharose 4B until 0.132 pmole of

vitamin B12 was present per milliliter packed volume. A column (20 ml)

was prepared and washed successively with 100 ml 0.1M K2HPO4, pH 7.5;

100 ml O.M glycine, 0.lM glucose, 1.OM NaC1, pH 10.0; then with 100 ml

7.5M guanidine HC1, 0.1M K2HP04, pH 7.5; and finally with 200 ml 0.1M





20
K2HPO4, pH 7.50. Immediately after washing, the IF fraction eluted from

the hydroxyl apatite column was applied by gravity flow. After sample

application the column was eluted with a linear 3.0 liter gradient (1.5

liters O.IOM K2HPO4, pH 7.50 1.5 liters 0.10M K2HPO4, 4.01 guanidine

HC1, pH 7.50) followed by flushing with 7.5M guanidine HC1, 0.10M K2HPO4,

pH 7.50. The material eluted in the gradient fractionation was 97 99%

IF as determined by the antibody inhibition assay. This material was dia-

lyzed, concentrated using an Amicon PM-10 ultrafilter, and stored frozen

at -70C until further use. The specific activity of this material was

11.5 ug vitamin B12 bound/mg protein using the charcoal binding assay and

29.8 ug vitamin B12 bound/ng protein as determined by vitamin B12 content

following complexation and gel filtration over G-25 Sephadex.

One step purification of IF and NIF by vitamin B12-Sepharose affinity

chromatograrn'v. Hog IF (25 gms)(l1x) was prepared as in the first vita-

min B12-Sepharose affinity ch'-omatogr&phy section to produce a celite fil-

tered crude extract. This was applied directly to the vitamin B12-Seph-

arose column (containing 1.32 pmole vitamin B12/ml) which was washed imme-

diately prior to use to remove any free vitamin B12. The column was washed

successively with 500 ml 0.:1, K2HP04, pH 7.5; then 1.0 liter 0.1M glycine,

0.1M glucose, 1.OM NaCl, pH 10.0; and lastly 500 ml 0.1M K2HP04, pH 7.50.

A linear 4.0 liter gradient (2.0 liters 0.1M K2HP04, pH 7.5 2.0 liters

4.0M guanidine HC1, 0.l'1 K2HP04, pH 7.50) was then passed through the

column followed immediately by a linear 2.0 liter gradient (1 liter 4.0M

guanidine HC1 1 liter 7.5M guanidine HC1 both in 0.10M K2HP04, pH 7.50).

The two vitamin B12 binding fractions that eluted were dialyzed, concen-

trated with an Amicon PM1-10 ultrafilter and stored at -700C.

Assessment of purity for IF and NIF. The homogeneity of each protein





21
fraction was determined by 7.5% polyacrylamide disc gel electrophoresis

and 7.5% polyacrylamide sodium dodecyl sulfate (SDS) disc gel electro-

phoresis (Appendix IV). Standard gels were run at 3 mamp per gel in a

Tris-glycine buffer at pH 8.2. Samples for SDS gel electrophoresis were

prepared by boiling each protein for 15 min. in 0.1% 2-mercaptoethanol

containing 1.5 pg SDS/pg protein. Gels were stained with Coumassie Bril-

liant Blue R-250 and scanned in a Gilford model 2400 spectrophotometer

equipped with a gel scanning apparatus.

The separation of IF from NIF was assessed by two methods. First,

each sample was subjected to the antibody inhibition assay. Secondly,

patient W.H. with documented pernicious anemia was given a Schilling Test

(Appendix V) using IF in one case and NIF in the other. IF (54 ug) was

completed with 500 ng [57Co]cyanocobalamin and given to U.H. The excre-

tion of [57Co]cyanocobalamin in the urine during 24 hrs. was then deter-

mined by counting the radioactivity in the urine using a Nuclear of Chi-

cago gamma counter. Similarly, NIF (76 pg) was completed with 500 ng

[57Co]cyanocobalamin and given to W.H. The excretion in the urine during

24 hrs. was assessed in an identical manner.

Trypsin Treatment of the Vitamin B12 Binding Proteins

as Used in all Subsequent Studies

Immobilized trypsin (200 mg) was suspended in 10 ml glass distilled

water and washed 10 times to remove buffer salts and any free trypsin.

An aliquot of this suspension, containing 50 mg of immobilized trypsin,

was assayed for tryptic activity using the benzoyl-DL-arginine-p-nitro-

anilide HC1 substrate as described in Appendix VI (41). This assay re-

vealed the presence of proteolytic activity equivalent to 14 pg crystal-

line bovine trypsin (Appendix VI).






22
Two hundred pg of each vitamin B12 binding protein (IF, NIF and Fx4)

was added to a plastic tube containing 50 mg of immobilized trypsin.

After adjusting the pH to 7.0 the tubes were incubated for 16 hrs. at

370C in a shaking water bath.

Following incubation, each tube was centrifuged in a clinical centri-

fuge at 3,000 RPM for 30 min. The supernatant solutions were removed by

aspiration and an aliquot of each supernatant solution was assayed for

tryptic activity using the assay system described in Appendix 'I.

The amount of proteolytic activity in each case was converted to

units of crystalline trypsin (ng) and revealed the presence of 3.3 ng in

the trypsin treated IF (TIF) fraction; 3.1 ng in the trypsin treated NIF

(TNIF) fraction; and 7.6 ng in the trypsin treated fraction 4 (TFx4)

material. The remainder of each fraction was stored at -700C until fur-

ther use.

The Effect of Trypsin Treatment on the Physiological

Effectiveness of Purified Intrinsic Factor

Patient J.P. has pancreatic insufficiency. When given a standard

Schilling Test (Appendix V) containing vitamin B12 alone, she excreted

an abnormally low 5.4% of the administered dose in 24 hrs. Therefore,

she also has vitamin B12 malabsorption. When she was given 500 ng vita-

min B12 completed to crude hog IF, she also excreted only 5.3%. There-

fore, she has no response to IF alone. When, however, she received vita-

min B12 plus pancreatic extract, her excretion rose to a normal 10.8%.

She, therefore, responds to pancreatic extract.

Enough purified hog IF to bind 600 ng vitamin B12 was incubated with

500 ng [57Co]cyanocobalamin at 37C for 30 min. The preparation was

administered orally to J.P. to establish the baseline for purified hog IF.






23
After a three-day interval, the same dose of purified hog IF was in-

cubated with 400 mg washed immobilized trypsin at 370C for 30 min. (equi-

valent in total dose exposure to the trypsin treated IF used for the re-

mainder of this study). The trypsin treated fraction was centrifuged at

3,000 RPM in a clinical centrifuge for 30 min. An aliquot of the super-

natant solution was removed and assayed for tryptic activity as outlined

in Appendix VI. During a 10-minute incubation, no tryptic activity could

be demonstrated. The supernatant solution of trypsin treated IF was then

completed with 500 ng [57Co]cyanocobalamin for 15 min. at 370C and admin-

istered orally to patient J.P. This.was followed in 5 days by a control

experiment using vitamin B12 alone.

The Effect of Trypsin Treatment on the Interaction of

Vitamin B12 with the Vitamin B12 Binding Proteins

The specific activity of the vitamin B12 binding proteins before and

after trypsin treatment. Each vitamin B12 binding protein and its tryp-

sin treated analog were assayed for protein concentration by the method

of Lowry (42). They were then assayed for vitamin B12 binding activity

by the charcoal binding assay (40) (Appendix II). Lastly, the amount of

material present which was still immunologically reactive as IF was de-

termined by the antibody inhibition assay (40) (Appendix III).

The equilibrium constant for binding of vitamin BI2 to the vitamin Bl2

binding proteins. The equilibrium constant for the reaction IF + B12
= ; IF B12 was determined by two different methods. In the first,

the method of Hummel and Dreyer (43) was used. A column of G-25 Sephadex

(0.9 x 60 cm) was equilibrated at 250C with 0.01M K2HP04, 0.15M NaC1,

pH 7.40, containing 100 ng/ml [57Co]cyanocobalamin (0.01 iCi/pg). A

known quantity of the protein (determined by the Lowry protein assay) was






24
mixed with exactly 1.0 ml of the equilibrating buffer and applied to

the column. Fractions (0.493 ml) were collected and the radioactivity

in each counted in a Nuclear of Chicago gamma counter for either 10 min.

or 10,000 counts. Assuming a 1:1 stoichiometry for this reaction, and a

molecular weight of 45,200 Daltons for IF, the equilibrium constants

were calculated as outlined in Appendix VI. Care was taken to use albu-

min coated glassware so that the IF preparations were quantitatively

transferred to the column. When this was not done, the equilibrium con-

stant was falsely depressed.

The equilibrium constants were also calculated by equilibrium dia-

lysis. Each binding protein (1 to 1.5 Vg) was brought to a final volume

of 10.0 ml with 0.01M K2HP04, pH 7.0, containing 1.0 mg/ml bovine serum

albumin. This sample was then placed inside a dialysis bag secured to a

stoppered plastic tube. The dialysis bag was placed in a sealed flask

containing 100 ml of the same buffer plus 150 ng [57Co]cyanocobalamin

(15 pCi/pg). As a control, the same experiment was conducted without

the protein inside the dialysis bag. Dialysis was continued for 304

hrs. at 25C in an oscillating water bath. At various time intervals,

samples (20 pi) were removed from both the outside and the inside of the

dialysis bag. After determining the total radioactivity in these sam-

ples, hemoglobin coated charcoal (2.0 ml) was added. After shaking for

15 min. and centrifugation in the clinical centrifuge (3000 RPM, 15 min.)

the radioactivity in an aliquot of the supernatant solution was counted.

The concentrations of free and bound vitamin B12 were then calculated

for each time point by difference.

At equilibrium, 1.0 ml of the solution inside and outside of the

bag was removed and the radioactivity measured to 10,000 counts. The






25
equilibrium constants were calculated from these data as outlined in

Appendix VII.

Kinetics of attachment of vitamin B12 to the vitamin B12 binding proteins.

The kinetics of the interaction of vitamin 812 with each 812 binding pro-

tein and its trypsin treated analog were determined as described by

Wagstaff et al.(ll). Both the forward and reverse reactions were studied.

A stock solution of 7.38 x 10-10M [57Co]cyanocobalamin (100 iCi/pg)

was prepared in 0.01M K2HPO4, pH 7.0, containing 1.0 mg/ml bovine serum

albumin. To determine the amount of vitamin 812 binding protein re-

quired to be equimolar with this concentration of vitamin B12, it was

necessary to perform the standard vitamin 812 binding assay using the

above 7.38 x 010-0M [57Co]cyanocobalamin solution. This was accomplished

by incubating (180 min. 25C) increasing amounts of protein with a stan-

dard amount of vitamin B12 followed by quenching with 0.2 ml hemnolobin

coated charcoal prepared in 0.01M K2HPO4, pH 7.0, containing 1.0 mg/ml

bovine serum albumin. From each binding curve was selected the amount of

binding protein strictly equimolar to the amount of vitamin B12 used in

the kinetic determinations.

For the reaction IF + B12 k-- IF 812, the second order rate con-

stant for the forward reaction, k1 can be directly determined under con-

ditions where [IF] and [B12] are initially equal. Reaction mixtures were

prepared by placing 1.6 ml phosphate buffer (0.01M K2HPO4, pH 7.0, con-

taining 1.0 mg/ml bovine serum albumin) plus 0.10 ml of 7.38 x 10-10M

vitamin B12 binding protein in glass tubes followed by preequilibration

for 5 min. at 250C in a water bath. Reactions were then initiated by the

addition of 0.10 ml of 7.38 x 10-10M [57Co]cyanocobalamin followed by mix-

ing with a Vortex mixer. Reactions were allowed to proceed at 250C for





26
the appropriate time intervals (10 600 sec.)followed by quenching with

0.2 ml hemoglobin coated charcoal in 0.01M K2'rPO4, pH 7.0, containing 1.0

mg/ml bovine serum albumin. After further incubation for 15 min., the

quenched reaction mixtures were centrifuged at 3,000 RPM. The radio-

activity in 1.0 ml of the supernatant solutions was counted for 10 min.

or to 10,000 counts. Similar determinations were performed using twice

the amount of both vitamin B12 and the binding proteins. Plots were con-

structed of 1/[B12free against time (sec.) and the second order rate

constants determined directly as the slope of these lines (Appendix VIII).

For the breakdown of the IF-B12 complex, the rate constant (k2) can

be directly determined from the relationship that Keq = kl/k2. Using the

apparent equilibrium constants derived in equilibrium dialysis experi-

ments in this buffer system, these rate constants were mathematically

determined.


The Effect of Trypsin Treatment on the Aggregation and

Conformation of the Vitamin B12 Binding Proteins

Polyacrylamide disc gel electrophoresis of the vitamin B12 binding pro-

teins and their trypsin treated analogs. The vitamin B12 binding pro-

teins (IF, NIF and Fx4), as well as their trypsin treated analogs (TIF,

TNIF and TFx4), were subjected to polyacrylamide disc gel electrophoresis

as described in Appendix IV. Each protein (10 pg) was run in triplicate

on the standard 7.5% polyacrylamide gel system at DH 8.2. The Rf values

were determined relative to Bromphenol Blue by scanning the gels in a

Gilford model 2400 spectrophotometer equipped with a gel scanning device.

The recorded peaks were triangulated and the centroid value was employed

for computation of the Rf value.

In the same fashion, sodium dodecyl sulfate (SDS) gel electro-





27
phoresis (Appendix IV) was performed in triplicate using 10 jg of each

protein. The Rf values were calculated in the same fashion.

Polyacrylamide disc gel electrophoresis of the vitamin B12 complexes of

the vitamin B12 binding proteins and their trypsin treated analogs.

Vitamin B12 complexes were prepared by incubating 0.2 ug of each protein

fraction with a 10-fold molar excess of [57Co]cyanocobalamin (100 pCi/pg)

for 8 hrs. at 250C. After incubation, the samples were dialyzed against

a 1000-fold excess of glass distilled water and subjected to polyacryla-

mide disc gel electrophoresis as outlined in Appendix IV. Each gel was

cut at the tracking dye front and sliced into 1 mm discs. The radio-

activity in each disc was then counted for 1 min. in a Nuclear of Chicago

gamma counter.

Sephadex chromatography of the vitamin B12 binding proteins. In an effort

to better understand the aggregational characteristics of IF-B12 and their

possible modification by trypsin treatment, these protein complexes were

subjected to reverse flow molecular sieve chromatography on both G-200

and G-150 Sephadex. A column of G-200 Sephadex (2.6 x 90 cm) was equili-

brated with 0.01, K2HP04, 0.751 NaCl, pH 7.50. Intrinsic factor was com-

plexed at 370C with excess [57Co]cyanocobalamin (15 pCi/pg) for a period

of 8 hrs. in the equilibrating buffer. This was followed by dialysis

against 1000 volumes of the equilibrating buffer to remove uncomplexed

vitamin B12. The complexes were cooled to 4C and applied to the column

using a flow rate of 15 ml/hr. Fractions were collected, weighed, and

their radioactivity counted to calculate the elution profile. The KAV

values were calculated from the relationship KAV = (Ve-Vo) / (Vt-Vo)

where Ve is the elution volume for the centroid position, Vo is the void

volume of the column determined using Dextran Blue 2000, and Vt is the





28
total bed volume. Separate experiments in which the protein concentra-

tion was varied from 0.094 pg to 30.0 pg IF were conducted to see if KAV

varied with concentration. An attempt was made to define the equilibrium

constant for aggregation by comparing the weight average molecular weight

with the protein concentration. Similar experiments were performed using

IF alone and TIF-B12.

Another reverse flow Sephadex G-150 column (2.6 x 90cm) was equili-

brated in the same buffer at a flow rate of 13 ml/hr. Intrinsic factor

(20 ug) was completed with excess [57Co]cyanocobalamin (15 pCi/pg) for

8 hrs. in the equilibrating buffer followed by dialysis. An identical

amount (10 pg) of NIF was prepared in the same fashion and these two prep-

arations were subjected to molecular sieve chromatography.

The Effect of Trypsin Treatment on the Interaction of the Intrinsic

Factor Vitamin 812 Complex with the Ileal Receptor

Preparation of the guinea pig ileal homogenate. Thirty male Hartley

guinea pigs (200 gm) were sacrificed by decapitation following a 12 hr.

fast. The small intestine was stripped from the mesentery from the

ileo-caecal junction to the ligament of Treitz. This was divided into

two equal parts and the distal half was flushed with 0.9% NaCl (50 ml)

at 4C. The mucosa was expressed with glass microscope slides and sus-

pended in 10 vols/wet weight of 0.14M NaCI, 0.005M KC1, 0.0025M CaC12,

0.00125M MgS04, 0.005M K2HPO4, pH 7.40 (Kreb's Ringer phosphate buffer

[KRP04]) at 40C. This mixture was homogenized for 30 sec. at full speed

in a Waring blendor, divided into 10 ml aliquots, and stored at -70C

until used.

Immediately prior to use, each aliquot was thawed at 40C and sus-





29

pended by approximately 10 strokes of a motor driven Teflon pestle. The

sample was centrifuged at 20,000 x G for 5 min. and the supernatant sol-

ution was decanted off and its volume measured. The pellet was washed

three times by suspension with a Vortex mixer in KRPO4 buffer lacking

calcium and magnesium. The washed pellet was finally suspended in a

volume of the same buffer equal to that of the initial supernatant sol-

ution.

Saturation of the vitamin B12 binding proteins with [57Co]cyanocobalamin

Purified hog IF and TIF were mixed with a 10-fold excess of [57Co]cyano-

cobalamin (100 lCi/pg) at 4C for 12 hrs. followed by dialysis against

1,000 volumes glass distilled water to remove unbound vitamin B12. The

amount of complex in each sample was determined by measurement of [57Co]-

cyanocobalamin. Each sample was diluted with KRPO4 buffer lacking cal-

cium and magnesium. The final concentration of each complex was 1,000 pg

(as vitamin B12) / ml.

Determination of the equilibrium constants for the binding of IF-B12 and

TIF-B12 complexes to the ileal receptor. The equilibrium constants for

the binding of IF-B12 and TIF-B12 to the guinea pig ileal homogenate were

determined at 250C using the standard guinea pig ileal homogenate assay

(44,45) described in Appendix X. The concentrations of the vitamin B12

complexes were varied from 1.0 pg (as B12) to 450 pg (as B12). Each re-

action mixture was incubated for 180 min. with a standard amount (0.20 ml)

of receptor homogenate. Incubation took place in 10 x 75 mm glass tubes

which had been soaked in bovine serum albumin for 2 hrs. and aspirated to

dryness. The reactions were quenched by filtration on 1.2 p millipore

filters which had been soaked in bovine serum albumin for 6 hrs. prior to

use. The amount of bound complex was determined by counting the radio-






30
activity on the millipore filters for 10 min. or 10,000 counts in a

Nuclear of Chicago garmma counter.

The equilibrium constants as well as the total number of receptor

sites available for binding were determined according to the method of

Steck and Wallach (46) (Appendix XI). Double reciprocal plots were con-

structed by plotting 1/[IF-B12]free against 1/[IF-Bl2]bound. The inter-

cept on the abscissa is related to the equilibrium constant as -Ka'

Similarly, the intercept on the ordinate (1/n) is related to the total

number of available binding sites (n) where n is expressed in units of

molarity.

The kinetics of attachment of IF-B12 and TIF-B12 to the guinea Dig ileal

receptor homogenate. For the binding of IF-B12 to the ileal receptor

site (IF-Bl2free + Receptorfree IF-B12bound), the reaction should
'be second order in the forward direction having a forward rate constant,

k1. To define this rate constant for the forward reaction, conditions

were adjusted so that the concentration of receptor sites at the start of

the reaction was equal to the concentration of IF-B12 in the incubation

mixture. Under these equimolar conditions, the rate law for formation of

the complex simplifies in a fashion identical to that of the binding of

vitamin B12 by IF (Appendix IX).

To define that concentration of IF-B12 exactly equal to the concen-

tration of receptor sites present in the incubation mixture, the values

of n calculated from double reciprocal plots for the equilibrium binding

of IF-B12 to the guinea pig ileal homogenate were employed. For IF-B12,

1/n = 2.2 x 1010M-1 and for TIF-B12, 1/n = 2.3 x 101M"1. For the stan-

dard reaction mixture of 1.0 ml this defines the concentration of receptor

sites available as 61.7 pg (as vitamin B12) for IF-B12, and 58.9 pg (as





31
vitamin B12) for TIF-B12. The average of these determinations (60 pg as

vitamin B12) was used for both IF-B12 and TIF-B12 in the experimental de-

termination of the forward rate constant.

The standard guinea pig ileal homogenate assay (Appendix X) was de-

vised for both IF-B12 and TIF-B12 with the concentrations of IF-B12 and

TIF-B12 set at 60 pg (as vitamin R12). Each kinetic experiment involved

a total of 10 determina.ions with incubation times ranging from 2 min. to

20 min. All reaction mixtures minus the vitamin B12 complex were pre-

pared and preequilibrated for 15 min. in a water bath controlled at 250C.

The reactions were then initiated by the addition of the vitamin B12 pro-

tein complex. The reactions were quenched at the end of the appropriate

time interval by rapid filtration on 1.2 p millipore filters presoaked in

bovine serum albumin. The millipore filters were then removed and the

radioactivity on each determined using a Nuclear of Chicago gamma counter

for 10 min. or 10,000 counts.

As described in Appendix IX for the binding of vitamin B12 to IF,

1/[IF-Bl2]free was plotted as a function of time. The slope of this line

represents the second order rate constant for the forward reaction. All

reactions were performed in duplicate and the average values utilized.

The first order rate constant for the dissociation of the IF-B12

receptor complex was then calculated directly from kI and Keq using the

relationship, k2 = kl/Keq.


The Effect of '!euraminidase Treatment on Intrinsic

Factor and Trypsin Treated Intrinsic Factor

Neuraminidase treatment of IF and TIF Neuraminidase (Clostridium per-

fringens) inmobilized on agarose beads was washed 10 times to remove buf-

fer salts and any free neuraminidase. It was then assayed for neuramini-





32
dase activity using bovine sulrmaxillary mucin (47) as outlined in Appendix

XII. Likewise, an aliquot was assayed using alpha-casein for total pro-

tease activity (48) as outlined in Appendix XIII. The neuraminidase acti-

vity was 7.0 x 10-5 units/mg and the protease activity was 4 x 10-6

units/mg under the incubation conditions.

For the treatment of IF and TIF, two different systems were employed.

In the first, 11 ng (as B12 binding activity) of each protein were dis-

solved in 1.0 ml 0.05M sodium acetate, 0.15M NaC1, pH 5.6, containing 2 mg

immobilized neuraminidase (1.4 x 10-4 units). Control preparations were

made in the identical fashion except that no neuraminidase was added.

The four reaction mixtures were placed inside dialysis bags and contin-

uously dialyzed for 16 hrs. against 10 ml 0.05M sodium acetate, 0.15M NaC1,

pH 5.6. Temperature was maintained at 370C in a shaking water bath. At

the end of the incubation period, all preparations were centrifuged at

20,000 x G for 5 min. and the supernatant solutions reiioved by aspiration.

In the second preparation scheme, 4 pg of each protein (inside an

albumin coated glass tube) was mixed with 1.0 ml 0.051 sodium acetate,

0.15f4 NaC1, pH 5.6, containing 2 mg immobilized neuraminidase. Control

samples were prepared in the identical fashion, except no neuraminidase

was added. The four samples were incubated in an oscillating water bath

at 370C for 16 hrs. At the end of the incubation, all samples were centri-

fuged at 20,090 x G for 5 min. and the supernatant solutions were removed

by aspiration.

Aliquots of the neuraminidase treated materials, and their controls

from both experiments, were then dialyzed against 1000 volumes of glass

distilled water, completed with a 5 fold-excess of [57Co]cyanocobalamin

(100 PCi/pg) for 24 hrs. and then dialyzed against distilled water for an






33
additional 24 hrs. These vitamin B12 completed fractions were used for

polyacrylamide disc gel electrophoresis and receptor binding studies.

Assessment of the vitamin B12 binding activity of neuraminidase treated

IF and TIF. The vitamin B12 binding activity of these four preparations

(from both experiments) was assayed using the charcoal binding assay

(Appendix II). The percentage of this material still immunologically

recognizable as IF was determined using the antibody inhibition assay

(Appendix III).

Polyacrylamide disc gel electrophoresis. The vitamin B12 complexes of

all four fractions derived from both sets of experiments were subjected

to polyacrylamide disc gel electrophoresis on a standard 7.5% polyacryla-

mide gel system run at 3 mamp/gel (Appendix IV). The gels were cut at

the tracking dye and sliced with a disc gel slicer. The radioactivity

in each disc was then counted for 1 min. in a Nuclear of Chicago gamma

counter.

Binding of the neuraminidase treated IF and TIF fractions to the guinea

pig ileal homogenate. The ability of the neuraminidase treated fractions

and their controls to bind to the guinea pig ileal receptor was measured

using the standard guinea pig ileal receptor homogenate assay (Appendix X).


The Effect of Neuraminidase Treatment of the

Guinea Pig Ileal Receptor Homogenate

Treatment with neuraminidase. Guinea pig ileal homogenate was prepared

as outlined in the previous section. An aliquot containing 10 ml of sus-

pension was centrifuged at 20,000 x G for 5 min. and the pellet stored in

ice. To this homogenized pellet was then added 3 ml 0.05M sodium acetate,





34
0.15M NaCI, pH 5.6, containing 3.0 mg neuraminidase (Type VI chromato-

graphically purified) which had been assayed for both neuraminidase

activity (7.2 x 10-3 units) and protease activity (4 x in-5 units) as

outlined in Appendices XII and XIII. A control incubation mixture was

prepared in an identical fashion except that no neuraminidase was added

with the buffer.

Both the control and neuraminidase treated homogenates were allowed

to incubate in a shaking water bath at 370C for 8 hrs. and were then

centrifuged at 20,000 x G for 5 min. The supernatant solutions were dis-

carded and each pellet was washed 3 times using 10 ml KRP04 buffer lack-

ing calcium or magnesium. The pellets were then resuspended to their

original volume using this same buffer.

The binding of IF-B12 to the neuraminidase treated receptor. The ability

of each preparation to bind to the IF-B12 complex was then measured using

the standard guinea pig ileal homogenate assay system as outlined in

Appendix X. Likewise, the ability of both preparations to bind TIF-B12

and neuraminidase treated IF and TIF completed to vitamin B12 was measured.


The Effect of Trypsin and Neuraminidase Treatment of Intrinsic

Factor on the Transport of Vitamin B12 Across the Ileal Cell

Preparation of the ileal gut sacs. Male Hartley guinea pigs (200 gms)

were fasted for 12 hrs. and sacrificed by decapitation. The terminal 10

cm of ileum was rejected and flushed with 20 ml KRPO4 buffer. Each ter-

minal ileum was then sliced into 3 slices, each of approximately 3 cm

length. These sections were pooled and stored in KRPO4 buffer until used.

Preparation of the vitamin B12 complexes of each binding protein. Puri-

fied intrinsic factor, trypsin treated intrinsic factor, neuraminidase





35
treated IF and TIF as well as the control, NIF, were incubated with a 10-

fold excess of [57Co]cyanocobalamin (100 pCi/mg) for 8 hrs. and were then

dialyzed against 1000 volumes of glass distilled water. These vitamin

812 completed proteins were used without further treatment.

Measurement of the transport of vitamin 812 across the ileal cell. For

each vitamin B12 completed protein, 3 gut sacs were used to determine the

amount of [57Co]cyanocobalamin transported across the ileal cell. Each

gut sac was removed from storage and weighed. The sac was then everted

onto a glass rod and one end was ligated with a silk suture. A blunt

needle was inserted through the open end of the sac and secured with a

ligature. Through the needle was then infused 0.5 ml buffer solution

(0.14M NaC1, 0.005M KC1, 0.0007M K2HP04, 0.0056r glucose, 0.025M tris-

hydroxymethylaminomethane, 0.0051 CaC12, pH 7.10, containing 50,000 units

penicillin per liter and 0.008 gm phenol red per liter). Each gut sac

was then suspended in 7.0 ml of this same buffer solution containing 1.0

ng of [57Co]cyanocobalamin completed to each of the various binding pro-

teins. The flasks were sealed and placed into an oscillating water bath

at 37C for 2 hrs. At the end of this time, the pH indicator dye start-

ed to change color and the incubations were stopped. Each ileal sac was

removed and rinsed by dipping into 3 sequential beakers containing 100

ml KRPO4 buffer. The sacs were dried by careful manipulation with filter

paper and each sac was opened with a longitudinal incision and its con-

tents expressed using gentle pressure with a pair of iris forceps. The

radioactivity inside each sac was counted using a Nuclear of Chicago

gamma counter.

The pH of each incubation mixture was measured and all were 6.9 at

the termination of the experiment. In several cases, the ileal sacs,






36
which were initially distended, had deflated during the incubation period.

This was taken as evidence of a leak and the results were not tabulated.

The amount of [57Co]cyanocobalamin transported was expressed as ng

[57Co]cyanocobalamin inside the sac per mg tissue/total ng [57Co]cyano-

cobalamin present at the initiation of the experiment.












RESULTS


Preparation of Vitamin B12-Sepharose Affinity Column


Isolation of vitamin B12 carboxylic acid derivatives. The acid hydro-

lyzed vitamin B12 was fractionated by successive chromatography on AG1-X8

anion exchange resin and QAE-A50 Sephadex. Figure III shows the results

of this latter chromatographic procedure. Titration of the material in

peak two indicated the presence of 1.19 moles carboxylic acid groups per

mole of vitamin B12. High voltage paper electrophoresis of this material

revealed three radioactive spots, one at the origin and two towards the

positive electrode. These were eluted and their radioactivity was counted.

Of the sample electrophoresed, 24% remained at the origin, 65% migrated

in a slow moving spot and 11% in a faster moving spot. This, combined

with the titration data, was consistent with the presence of 24% vitamin

Bl2, 65% monocarboxylic acid and 11% dicarboxylic acid derivatives of

vitamin B12 (49,50,51,52).

Preparation of 1,6-hexanediamine linked Sepharose 4B. Sepharose 4B was

covalently linked to 1,6-hexanediamine using CNBr activation. Following

extensive washing, an aliquot (1.0 ml) of this slurry was titrated with

1 x 10-2M HC1 to reveal 17 pmole titratable amine per ml packed column

material.

Coupling with vitamin 812 carboxylic acid derivatives. The vitamin B12

monocarboxylic acid derivative was linked with a water soluble carbodi-

imide to 1,6-hexanediamine substituted Sepharose. Following extensive

37


















CO
.0


*C0





1000 2000 3000 4000
EFFLUENT, L .
FIGURE III Chromatography of the Eluate from AG1-X8 Ion Exchange

Chromatography on QAE-A50 Sephadex

A column (2.5 x 80) of QAE-A50 Sephadex was poured and equilibrated

in 0.2M pyridine (pH 9.0 with ammonium hydroxide). The AG1-X8

eluate was applied in 0.2M pyridine (pH 9.0 with ammonium hydroxide)

and was followed by 1500 ml 0.4M pyridine. The column was then eluted

with a 2.0 liter linear gradient from 0.4M pyridine to 0.4M pyridine

with 0.16M acetic acid. Finally, the column was flushed with 1.0

liter 0.4M pyridine with 0.32M acetic acid. The flow rate was 15

ml/hr.and the chromatography was performed at 40C.






39
washing, an aliquot (1.0 ml packed volume) of Sepharose was counted in a

Nuclear of Chicago gamma counter indicating 1.32 pmole vitamin B12 cou-

pled per ml Sepharose.


Purification of Hon IF and NIF

First vitamin B12-Sepharose affinity column. Figure IV shows the elution

profile for this first purification step. A total of three protein peaks

were eluted, only one of which (that appearing in O.IM K2HPO4, 7.5M gua-

nidine HC1, pH 7.5) had any demonstrable vitamin B12 binding activity.

In this single passage over the affinity matrix, 60% of the vitamin B12

binding activity and 5% of the total protein applied were recovered.

This represents an overall purification of 114-fold and yields a prepara-

tion that binds 2.97 vg vitamin B12 per mg protein as determined in the

charcoal binding assay and the Lowry protein assay (42).

Second vitamin B12-Sepharose affinity column. The elution profile for

this purification step is shown in Figure V. All the vitamin 812 binding

activity was contained in the guanidine HC1 washings. The antibody inhi-

bition assay indicated that the material eluted in 7.5M guanidine HC1 was

99% NIF. This material represents a recovery of 34% of the vitamin B12

binding activity originally present in the crude extract as NIF and

yields an overall purification of 58-fold. Similarly, the material eluted

with guanidine HC1 ranging from 3.0 to 6.OM contained vitamin 812 binding

activity which was 70% IF and 30% NIF. This represents an overall recov-

ery of 10% of the vitamin B12 binding activity originally present in the

crude extract as IF and yields an overall purification of 35-fold.

Hydroxyl apatite chromatography of NIF. The material isolated from the

7.5M guanidine HC1 washing of the second affinity column was further puri-

fied with hydroxyl apatite. The elution profile for this step is seen in







40





O

C
0
C \J



0 o ^

V.. ... gL^..
1000 3000 5000 *7000 9000
EFFLUENT, ML .
FICPUJr IV Chromatography of Crude Hog IF on the First Vitamin Bi12-
Sepharose Affinity Column
A column (50 ml) of vitamin B12-Sepharose (1.32 pmole B12/ml) was
equilibrated at 40C. with 0.1M K2HP04, pH 7.50. The crude IF homog-
enate was applied in 0.1M Tris-acetate, pH 9.2 at a flow rate of
15 ml/hr. The column was successively eluted with .1.0 liter O.lM
K2HPO4,'pH 7.50; 2.0 liter 0.lM glycine, l.OM NaC1, pH 10.0; 1.0
liter O.IM K2HP04, pH 7.50; and finally with 1.0 liter 7.5M
guanidine HC1 in 0.1M K2HP04, pH 7.50. (o), A280; (x), vitamin B12
binding activity (pg/ml).













E

E- --I
0
CO

SL'l
co







1000 2000 3000 4000
EFFLUENT, ML.


FIG.U.E V The Secord Vitamin B!2-Spharose Affinity Colu-n

The vitamin B12-Sepharose affinity column (50 ml) was pre-

pared in the same "-rner as the first vitanin B12-Sepharose

affinity column. The fraction isolated from the first af-

finity column which contained all the vitamin 812 binding

activity was applied at 40C at a flow rate of 15 ml/hr.

The column was successively \;ashed with 500 ml 0.Ill K2HPO4,

pH 7.50; 600 ml 0.1il glycine, I.OM NaCl, oH 10.0 ar.d 400 ml

0.1M K2HP04, pH 7.50. A series of buffers (200 ml each) pre-

pared in 0.1M K2HPO4, pH 7.50 were then used to differen-

tially elute IF and NIF. These buffers contained guanidine

HCl at concentrations of 1, 2, 3, 4, .5, 6, and 7.5M. (&),

,A280; (x), vitamin B12 binding activity (pg/ml).





42

Figure VI. Two vitamin B12 binding peaks were evident. The first was

rechromatographed to a constant specific activity of 9.50 pg vitamin B12

bound/mg protein using the charcoal binding assay. This represents a

365-fold purification of NIF with a 15% recovery. The total yield was

200 pg vitamin B12 binding activity and 21 m:a protein. This material

(32 pg) was' completed with 1.0 pg [57Co]cyanocobalamin for 16 hrs. at 4C

and chromatographed over G-25 Seohadex. The specific activity measured

in this fashion was 17 pg vitamin B12 bound/mg protein on the final prep-

aration. The second peak, which elutes at much higher ionic strength,

is referred to as Fx4 and represents 12 pg vitamin B12 binding activity

and 8.4 mg protein. Its specific activity was 1.4 pg vitamin B12

bound/mg protein.

Hydroxyl apatite chromatography of IF. The material pooled in the 3.0 to

6.0M guanidine HC1 washes of the second affinity column was also chromato-

graphed on hydroxyl apatite. The first peak of vitamin B12 binding activ-

ity represented 49 pg vitamin B12 binding activity which was 65% IF.

This material had a constant specific activity of 7.0 pg vitamin B12

bound/mg protein and represented a recovery of 15% of the vitamin B12

binding activity originally present in the crude extract as IF as well as

an overall 269-fold purification.

Third vitamin B12-Sepharose affinity column. The IF preparation obtained

from hydroxyl apatite chromatography was subjected to further vitamin B12

affinity chromatography using a linear guanidine HC1 gradient. Figure

VII illustrates that two peaks of vitamin B12 binding activity were ob-

tained. The first of these was 99% IF as determined by the antibody in-

hibition assay and represented a total of 10% of the IF present in the

crude extract with an overall 430-fold purification. The specific activ-















I\,









1000 2000 -000 4000
EFFLUENT, ML

FIGURE VI Hydroxyl Apatite Chromatography of NIF
A colunn (2.5 x 30 cm) of hydroxyl apatite was prepared
in distilled water. The fraction eluted in 7.5M guan-
idine HCI, 0.1M[ K2HPO4, pH 7.50 during the second vita-

flow at 4'. A 2.0 liter linear gradient was run from

distilled water to 0.05:1 K2HP04, pH 7.0; followed by. a
1.0 liter linear gradient from 0.05 to 1.0' K2HPO4,
pH 7.0. (o), A280; (x), vitamin 812 binding activity
(vg/ml).












LL.

-0.




cr) r) E
j / \
0QN" / X
x 0

O100 / x3 I 4o
< \ I 1
0 x/ z /I




EFFLUENT, ML.

FIGURE VII The Separation of IF from NIF on the Third Vitamin B1?-
Sepharose Affinity Column

A 20 ml column (0.132 pmole B12/ml) of vitamin B12-Sepharose was

washed in 0.1M K2HP04, pH 7.50. The fractions eluted between 3 and

6M guanidine HC1, 0.11 K2HPO4, pH 7.50 during the second vitamin B12

affinity column (IF and NIF) were applied by gravity flow. The column

was eluted with a 3.0 liter linear gradient from 0.1M K2HP04, pH 7.50,

to 4.OM guanidine HC1, 0.1M K2HP04, pH 7.50. The column was then

flushed with 7.50M guanidine HC1, 0.1M K2HP04, pH 7.50. (o), A280;

(x), vitamin B12 binding activity (pg/ml); (e), sensitivity to
anti-IF antibody (%IF).





45
ity was 11.2 pg vitamin B12 bound/mg protein as measured in the charcoal

binding assay. This same material (9.4 yg) was completed with 500 ng

[57Co]cyanocobalamin at 4C for 16 hrs. The complex was then passed over

G 25 Sephadex and the vitamin B12 content of this preparation yielded a

specific activity of 29.8 pg vitamin B12 bound/mg protein. The overall

purification scheme and the results at each stane are outlined in Table 1.

One-step purification of IF and NIF by vitamin B12-Sepharose affinity

chromatography. Figure VIII outlines the elution profile obtained using

this one step chromatographic method. The first peak of vitamin Bl2 bind-

ing activity, formed by pooling the 99% to 97% IF (as determined by anti-

body inhibition assay) fractions, yielded 143 pg vitamin B12 binding

activity with a specific activity of 4.6 ig vitamin B12 bound/mg protein.

This represents a recovery of 85% of the IF originally present in the

crude extract. Similarly, the second peak of vitamin B12 binding acti-

vity contained 930 pg vitamin B12 binding activity of a specific activity

equal to 7.8 pg vitamin 812 bound/mg protein. This represents a recovery

of 82% of the NIF originally present in the crude extract. It was 92%

NIF as determined by the antibody inhibition assay. Table 2 summarizes

these overall results.

Assessment of purity for IF and NIF. Polyacrylamide disc gel electro-

phoresis of IF, NIF and Fx4 was performed in triplicate. The average Rf

values are found in Table 3. Most importantly, a single band was found

for both IF and NIF. Likewise, the SDS gel electrophoresis yielded one

band for IF and one for NIF. These facts suggest that these two proteins

were horlogenous. The third vitamin 812 binding protein, Fx4, exhibited

two bands on regular polyacrylamide gel electrophoresis and a single band

on SDS gel electrophoresis. It is likely that this material was either



















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2000 4000 6000 8000
EFFLUENT, ML.

FIGURE VIII One-Step Procedure for the Purification of IF and NIF

A 50 ml column (1.32 role Bl2/ml) of vitamin B12-Sepharose was
washed with 0.1M K2HPO4, pH 7.50. The crude extract from 25 gm of

lOx IF was applied and fractionated by successive elution with 500 ml
O.M K2HPO4, pH 7.50; 1.0 liter 0.lIAlycine, F.01 NaCI,'pH 10.0;

500 ml 0.M K2HPO, pH 7.50; a 4.0 liter linear gradient from 0.IM
./\ % c
















K2HPO4, pH 7.50 to 4.0M guanidine HCl, 0.lMK2HPO4, pH 7.50 and
finally, a 2.0 liter linear gradient from 4.0 to 7.5M guanidine HCl
in K2HP4, p 7.50. (o), A280; (x),vitamin 2 binding ac-

tivity (00g/m); (), sensitivity to anti-IF antibody (F).
EFFLUENT, ML.

FIGURE VIII One-Step Procedure for the Purification of IF and NIF
A 50 ml column (1.32 mole B12/ml) of vitamin B12 -Sepharose was
washed with O.AM K2HP04, pH 7.50. The crude extract from 25 gm of
lOx IF was anplied anO fractionated by successive elution with 500 ml
0.1M K2HP04, pH 7.50; 1.0 liter 0.AM glycine, I.0, NaCl,'pH 10.0;
500 ml 0.1AMK2HP04, pH 7.50; a 4.0 liter linear gradient from O.AM
K2HP04, pH 7.50 to 4.OM guanidine HC1, 0.AM K2HP04, pH 7.50 and
finally, a 2.0 liter linear gradient from 4.0 to 7.5M guanidine HC1
in O.M K2HP04, pH 7.50. (o), A280; (x),'vitamin B12 binding ac-
tivity (pg/ml); (e), sensitivity to anti-IF antibody (:IF).





















S-
0
4-)

Li
u



ro
Lt
S-
4-)
C




t-z


ro
0








Lt-
En


0




5-

t4-


Lo




S 0










.-r-
4c-

4---
0-



C


C)
C-







S0
Ei n




4)

Li
(/)





0

.3-
0-

a-




C



4-)

(4
0
U)


a)


c
0
4r-
Cr LO CY)
U C
4-



CL
S-












0.
S--


Cu
> 0C LO CM

u L i
>)




(0


4- *-
*r- a)






4- c4
*r- 0
4-) S.-
O 0Q. CO U CO

cn 'd"- g~
u Ec
.5- CM








a




CN
CL 01 .







C O





.-
CD
rQ --- -----------



.S-






C 0







*r- LI r- ci
4- E eO-
o ,
a-




4..


r- S.-

*r I X
S.. LLI Li.. Li..

0 -0
"E 3
(_>


to -
.0 p
.*r- 0
4-> -


0

0 -0
o o

0 4-)
4- a)
E
Li

C4--

4-C 3
*r-

4-) .0
C
*- -0
a)
*r- C
-> *r-
C E
>a >

0 5-



*- (0

*- C
> *r-
r- ao
U 4.)
C 0
u sa.


0 L
- '- -


>r
0
tI


eni
E,
v5
-o
CD

*r-
*r
i-

.0
0
Li

0


a
-c


.C




-o,
a)
C
.Q..
*--
E
L.
5-
a)
4-)



(/I

r3


*r-
>0
*r-
0

*r-
4-
*r-

0-









Results of

of


TABLE 3

Polyacrylamide Disc Gel Electrophoresis

the Vitamin B12 Binding Proteins


(a) Standard error of the mean


Sample # Bands R S.E.M.(a)

IF 1 0.34 0.01

NIF 1 0.29 0.008

Fx4 2 0.19 0.014, 0.29 0.015

IF SDS 1 0.58 0.007

NIF SDS 1 0.43 0.012

Fx4 SDS 1 0.44 0.018





50
contaminated with another protein fraction or had some aggregational prop-

erty destroyed by SDS. Later, it was shown that Fx4 B12 complexes

electrophorese as two bands and it is possible that the two bands seen

here may both have vitamin 812 binding activity.

The fraction isolated as IF was greater than 99. inhibitable by anti-

IF antibody (blocking type) which would attest to its lack of contamina-

tion by NIF. Likewise, the Schilling test performed with 54 pg protein

had a normal, 12.2' excretion for the 24 hr. period. This indicates its

physiological effectiveness in all phases of the absorptive scheme. A

repeat test with 76 pg NIF had 1.2% excretion for 24 hrs. This was mark-

edly less than the normal, 8%, and indicates that NIF was not signifi-

cantly contaminated by IF. The antibody inhibition assay of NIF revealed

less than 1% inhibition which also supports the lack of IF in this prep-

aration. Fraction 4 (Fx4) was also less than 1% inhibitable by anti-IF

antibody.

The specific activity of IF as measured by vitamin B12 content (29.8

yg vitamin B12 bouirnd/mg protein) is very similar to the value of 30.3

pg/mg found by Allen and Mehlman (10). This number represents a 1:1

stoichiometry based on a molecular weight of 45,200 Daltons. The specif-

.ic activity of NIF (17 pg vitamin B12 bound/mg protein) is slightly less

than the value of 25.1 pg/mg found by Allen and Mehlman (10).


The Effect of Trypsin Treatment on the Physiological

Effectiveness of Purified Intrinsic Factor

Following administration of 500 ng vitamin B12 completed to purified

IF, J.P. excreted 7.3% of the total dose in the first 24 hrs. This was

abnormally low. However, using 500 ng vitamin B12 completed to trypsin

treated purified IF, J.P. excreted 18.4% of the total dose in the first





51
24 hrs. This was clearly within the normal range. The subsequent control

absorption, using vitamin B12 alone, was a basal 5.0%.


The Effect of Trypsin Treatment on the Interaction of

Vitamin B12 with the Vitamin B12 Binding Proteins

Specific activity of the vitamin D12 binding proteins before and after

trypsin treatment. The data for all three vitamin B12 binding proteins

before and after trypsin treatment are summarized in Table 4. For IF, the

amount of protein present, the specific activity in terms of vitamin B12

bound/mg protein, and the amount of vitamin B12 binding still sensitive

to anti-IF antibody are essentially the same. This indicates that no new

binding sites for vitamin B12 were exposed by trypsin treatment and that

the trypsin treated material was still structurally similar enough to

interact with the anti-IF antibody.

Trypsin treatment of NIF and Fx4 does not destroy their ability to

bind vitamin B12 nor does it convert these proteins into IF as evidenced

by the inability of anti-IF antibody to inhibit these preparations.

The equilibrium constants for vitamin Bl2 binding to the binding proteins.

The equilibrium constants for vitamin B12 binding to both pure IF and NIF

were measured by chromatography on G-25 Sephadex. Figure IX shows an

elution profile for such an experiment using purified hog IF. The equilib-

rium constants (Appendix VI) are 1.8 x 109M-1 and 0.2 x 109M-1 for IF

and NIF, respectively.

The equilibrium constants for vitamin B12 binding to all the vitamin

B12 binding proteins and their trypsin treated analogs were measured by
equilibrium dialysis. Two sets of equilibrium constants were calculated

from these data (Appendix VII). The first of these, the apparent equilib-

rium constant, is dependent only upon the amount of vitamin B12 binding






52

T.BLE 4

Specific Activity of the Vitamin 812 Binding

Proteins Before and After Trypsin Treatment


materiall Protein Vitamin B12 Binding Specific Activity

mg pg % IF (Ipg B12 bound/mg protein)

IF 0.174 2.2 99 12.6

NIF 0.200 1.6 1 8.0

Fx4 0.300 0.31 1 1.0

TIF 0.185 1.8 99 9.6

TNIF 0.284 1.7 3 6.2

TFx4 0.382 0.44 2 1.1





















.O
20




10 20 30 40 50 60 70 80 90
FRACTION NUMBER
FI D"E IX Determination of the Equilibrium Constant by Filtration
Over G-25 Seohadex for the Binding

of Purified Hog IF to Vitamin BI2
A column of G-25 Sephadex (0.9 x 60 cm) was equilibrated at 250C
with 0.01M K2HP04, 0.15M NaC1, pH 7.40, containing 100 ng/ml [57Co]-

cyanocobalFmin. Purified IF (9.4 pg) was incubated for 5 min. in
1.0 ml of this equilibrating buffer in an albumin coated glass tube.

The sample was applied and fractions (0.493 ml) were collected.
The radioactivity in each fraction was counted in a Nuclear of Chi-

cago garmT.a counter for either 10 min. or 10,000 counts.






54
activity present. No assumptions are made regarding the molecular weight

of the binding protein (Table 5). The second type of equilibrium con-

stant involves estimation of the protein's molecular weight. These

thermodynamic equilibrium constants (Table 6) and the values measured by

gel filtration are, within experimental error, approximately equal. This

suggests that these values approximate the true equilibrium constant and

are not affected by either the dialysis or the gel filtration system.

Trypsin treatment did not alter either the apparent or thermodynamic

equilibrium constants for vitamin B12 binding to any of the vitamin B12

binding proteins. The apparent equilibrium constants are very similar to

those obtained both by McGuigan (53) and by Allen and Mehlman (9,10).

Kinetics of attachment of vitamin Bl2 to the vitamin B12 binding pro-

teins. Figure X shows kinetic data for pure IF binding to vitamin B12.

The reaction exhibits strict second order kinetics and, since the slopes

obtained at two different concentrations of IF and vitamin B12 (3.69 x

10-11M and 7.33 x 10-11M) are essentially equal, there is no dependence

of this reaction on protein concentration. This rules out the effect of

polymerization under these experimental conditions. The second order

rate constants for vitamin B12 binding to all of the binding proteins

and their trypsin treated analogs are summarized in Table 7.

The rate constants for the dissociation of the vitamin B12 binding

protein complexes were calculated from the apparent equilibrium con-

stants and the second order rate constants for association. These data

are summarized in Table 8.

Trypsin treatment does not effect the rate constants for either for-

mation or dissociation of any of the vitamin B12 complexes. Thus, its

in vivo effect of facilitating vitamin B12 transport cannot be explained






55
TABLE 5

Apparent Equilibrium Constants for the Binding of

Vitamin B12 to the Purified Vitamin B12 Binding

Proteins and Their Trypsin Treated Analogs



Material Equilibrium Constant

( x 10-OM-')

IF 1.00

NIF 0.86

Fx4 1.00

TIF 0.94

TNIF 1.00

TFx4 1.30






56
TABLE 6

Thermodynamic Equilibrium Constants for the Binding of

Vitamin B12 to the Purified Vitamin 812 Binding

Proteins and Their Trypsin Treated Analogs


t1aterial Equilibrium Constant(a)

( x 10-9-)


IF 4.0

NIF 4.4

Fx4 1.6

TIF 3.0

TNIF 6.2

TFx4 1.0


(a) Assuming molecular weights of 65,000 Daltons

for both NIF and Fx4 as well as their trypsin

treated analogs. The molecular weight of both

IF and TIF is assumed to be 45,200 Daltons.














; --










100 200 300 400
TIME., SEC.

FIGURE X Kinetics of Attatchment of Vitamin B12 to Purified IF

Intrinsic factor and [57Co]cyanocobalamin (100 y Ci/g) were ad-

justed to equimolar concentrations. A series of reaction mix-
tures were set up in glass tubes (10 x 75 mm) at 250C. Each in-

cubation mixture contained 1.6 mi O.OlM_ K2HP04, pH 7.0, contain-

ing 1.0 mo/mi bovine serum albumin plus, 0.1 ml binding protein

solution (7.38 x 10-10M). These were incubated for 5 min. before
a stoichiometric amount of [57Co]cyanocobalamin was added. The

reactions were quenched at the appropriate time intervals by the

addition of 0.2 ml hemgcilobin coated charcoal prepared in the eq-
uilibrating buffer. The reactions were centrifuged and the radio-

activity in each suoernatant was counted. (x), reacting concen-

trations were 3.69 x 10-11[; '(o), reacting concentrations were

7.38 x 10-11M.
6^.' ^^
**- /-? ^^ X'-<






58
TABLE 7

Second Order Rate Constants for the Attachment

of Vitamin B12 to the Vitamin B12 Binding

Proteins and Their Trypsin Treated Analogs


(a) Molar concentration ot

was 3.69 x 10-11M

(b) Molar concentration of

was 7.38 x 10-11M


vitamin B12 and each binding protein


vitamin 812 and each binding protein


Material Second Order Rate Constant

( x 10-8M-1sec-1)

(3.69 x 10-1M!1)(a) (7.38 x 10-11M)(b)


IF 0.79 0.80

NIF 1.0 0.97

Fx4 0.18 0.32

TIF 0.71 0.85

TNiF 0.55 0.57

TFx4 0.32 0.37





59
TABLE 8

First Order Rate Constants for the Dissociation of the

Vitamin B12 Complexes with the Vitamin B12 Binding

Proteins and Their Trypsin Treated Analogs


Material First Order Rate Constant

( x 102sec-1)

IF 0.83

NIF 1.0

Fx4 0.25

TIF 0.84

TNIF 0.56

TFx4 0.27





60
by an alteration in the kinetics of the interaction with vitamin B12.


The Effect of Trypsin Treatment on the Conformation and

Aggregation of the Vitamin B12 Binding Proteins

Polyacrylamide disc gel electrophoresis of the vitamin B12 binding pro-

teins and their trypsin treated analogs. The Rf values, relative to the

migration of Brcmphenol Blue, obtained by polyacrylamide gel electro-

phoresis of each vitamin B12 binding protein and its trypsin treated ana-

log, are found in Table 9. Table 10 lists the same results for SDS gel

electrophoresis. The number of bands stained with Coumassie Brilliant

Blue R250 is also listed.

Intrinsic factor behaves as a single band both before and after

trypsin treatment. This is seen on both regular and SDS polyacrylamide

gel electrophoresis. Furthermore, the average Rf values for intrinsic

factor compared to trypsin treated intrinsic factor are not statistically

different. This would suggest that no appreciable changes in either

charge, size, or conformation occurred as a result of trypsin treatment.

Non-intrinsic factor also behaves as a single band both before and

after trypsin treatment. Likewise, the average Rf values for NIF com-

pared to TNIF are not statistically different.

Fraction 4 exhibits variable results. Before trypsin treatment,

there are 2 bands on regular gel electrophoresis which condense to one

band on SDS gel electrophoresis. After trypsin treatment, there is 1

band on regular gel electrophoresis and 2 bands on SDS gel electrophore-

sis. A consistent finding, however, is the presence of one of the bands

on regular gel electrophoresis both before and after trypsin treatment,

which has an Rf value of 0.285 0.015 before and 0.289 0.008 after






61
TABLE 9

Polyacrylamide Disc Gel Electrophoresis of the Vitamin

B12 Binding Proteins and Their Trypsin Treated Analogs


(a) Average of three


Average Rf Relative to
Material Bromphenol Blue(a) # of Bands

(+ S.E.M.)


IF 0.34 0.01 1

NIF 0.29 0.008 1

Fx4 0.19 0.014, 0.29 0.015 2

TIF 0,35 0.006 1

TNIF 0.29 0.015 1

TFx4 0.29 0.008 1





62
TABLE 10

Sodium Dodecyl Sulfate Polyacrylamide Disc Gel

Electrophoresis of the Vitamin B12 Bindir-

Proteins and Their Trypsin Treated Analogs


(a) Average of three


Average Rf Relative to
Material nol Blue(a # of Bands
Bromphenol Blue(a)

( S.E.M.)


IF 0.58 0.007 1

NIF 0.43 0.012 1

Fx4 0.44 0.018 1

TIF 0.56 0.01 1

TNIF 0.42 0.007 1

TFx4 0.22 0.01, U.47 0.01 2





63

trypsin treatment. Likewise, a band on SDS gel electrophoresis both

before (Rf = 0.440 0.018) and after (Rf = 0.469) trypsin treatment can

be found. The Rf values obtained for either regular or SDS gel electro-

phoresis are not statistically different from each other. Interestingly

enough, these values are also not statistically different from those ob-

tained for NIF. The relationship of the other bands to Fx4 remains, at

present, unclear.

Polyacrylamide disc gel electrophoresis of the vitamin Bl2 complexes with

the B12 binding proteins and their trypsin treated analogs. The gel pro-

files for each vitamin B12 completed protein and its trypsin treated ana-

log were plotted as mobility relative to Bromphenol Blue. Figure XI has

the profile for IF-B12 and TIF-B12. It can be seen that at least two dis-

tinct bands are present in each case. These bands have relative Rf

values of 0.27 and 0.45 for IF-B12 and 0.28 and 0.44 for TIF-B12. They

are essentially identical in mobility both before and after trypsin treat-

ment. Furthermore, the relative proportion of the first band to the sec-

ond remains constant. This would indicate the presence of two species

of vitamin B12 completed to both intrinsic factor and trypsin treated

intrinsic factor. Whether these species differ by charge or by size can

not be determined from these analyses, but, since IF and TIF gave single

bands on electrophoresis without vitamin B12 and since the vitamin B12

complex of intrinsic factor has been shown to aggregate into higher mole-

cular weight species (4,5,9, 10), it may indeed be that these two peaks

represent monomeric and polymeric states of the molecule. If this is

true, it seems likely that trypsin has no gross effect upon the ability

of IF-B12 to aggregate into polymeric forms.

The profiles shown in Figure XII are for NIF-B12 and TNIF-B12. Once























0.1 0.2 03 0.4 0.5 0.6 0.7 0.8 0.9
MOBILITY RELATIVE TO BROMPHENOL BLUE

FIGUFE XI Polyacrylnmide Disc Gel Electrophoresis of the

Vitamin B?2 Cor plexes of IF and TIF
The vitamin 812 ccorplexes of IF and TIF were prepared by in-
57C
cubating 0.2 ig of each p;r.tein with a 10-fcld excess of [5 Co]-
cya-ocobalamin for 8 hrs. at 250C. The cor.plexes were dialyzed

against 100O volumes of glass distilled water and subjected to

polyacrylamide disc eel electrophoresis on the standard 7.5%
polyacrylamide gel system described in Appendix IV. The gels

were sliced at 1 mm intervals and the radioactivity in each

disc was counted. (o), IF-B12 complex; (x), TIF-Bl2 complex.











0o
x0

x x





ktx.
0.41 .2 0.3 0.4 0.5 0.6 0.7 0.0 0.9
MOBILITY RELATIVE TO BROMPHENOL BLUE
FIGUPE XII Polyacrylamide Disc Gel Electrophoresis of the
Vitamin B12 Cormlexes of NIF and TNIF

The vitamin B12 complexes of I!IF and TNIF were prepared by in-
cubating 0.2 pg of each protein with a 10-fold excess of [57Co]-
cyanocobalamin for 8 hrs. at 250C. The complexes were dialyzed
against 1000 volumes of glass distilled water and subjected to
polyacrylamide disc gel electrophoresis on the standard 7.5%
polyacrylamide gel system described in Appendix IV. The gels
were sliced at 1 mm intervals and the radioactivity in each
disc'was counted. (o), NIF-B12 complex; (x), TNIF-B12 complex.





66

again, two species are present for both NIF-B12 (Rf = 0.22 and 0.36) and

TNIF-B12 (Rf = 0.21 and 0.37). The mobility of these two species does

not appear to be affected by trypsin treatment of NIF. However, it is

clear that the proportion of material present in the slower moving band

is decreased by trypsin treatment. Whether this represents a change in

molecular charge or size can not be determined by this experiment. Tryp-

sin is therefore capable, at the same dose level used to treat IF, of

modifying the structure of NIF to change the relative proportion of the

two electrophoretic bands.

Figure XIII depicts the electrophoretic profiles obtained for Fx4-

812 complexes. For both the trypsin treated and untreated material, two

poorly resolved but distinct bands of radioactivity are obtained. The Rf

values for these two bands neither change upon treatment with trypsin

(untreated Rf = 0.27 and 0.38; treated Rf = 0.28 and 0.38), nor is there

any change in the relative proportion of the two bands. It seems clear

that treatment with trypsin does not alter the mobility of the Fx4-B12

complex.

Sephadex chromatography of the vitamin Bl2 binding proteins. Table 11

lists the KAV values obtained from G-200 Sephadex chromatography of IF,

IF-B12 and TIF-B12. These results clearly indicate that KAV does not
change as a function of IF-B12 concentration. Since KAV is directly re-

lated to the weight average molecular weight, it is, therefore, not possi-

ble to quantitatively measure the formation of IF-B12 aggregates. The

KAV value for IF as well as that for TIF-B12,falls well within the range

of experimental error ( 2 S.D.) defined by the values of KAV for IF-B12.

The TIF-B12 chromatogram also demonstrates the same amount of aggregation

as does that of IF-B12.













O


t*-, I I;
0 0 X

x

d x' x

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
MOBILITY RELATIVE TO BROMPIHENOL BLUE

FIGURE XIII Polyacrylamide Disc Gel Electrophoresis of the

Vitamin B12 complexes of Fx4 and TFx4

The vitamin B12 "complexes of Fx4 and TFx4 were prepared by in-

cubating 0.2 pg of each protein with a 10-fold excess of [57Co]-

cyanocobalamin for 8 hrs. at 250C. The complexes were dialyzed

against 1000 volumes of glass distilled water and subjected to

polyacrylamide disc gel electrophoresis on the standard 7.5%
.polyacrylamide gel system described in Appendix IV. The gels

were sliced at 1 mm intervals and the radioactivity in each

disc'was counted. (o), Fx4-B12 complex; (x), TFx4-B12 complex.
____^ ^ ^________





68

TABLE 11

Molecular Sieve Chror-atography of the Intrinsic Factor

Vitamin B12 Complex Using G-200 Sephadex


Material Total Protein KAV
(pg)

IF 9.40 0.426(b)

IF-B12 0.094 0.453(a)

0.282 0.434

3.76 0.500

9.40 0.465

30.0 0.461

TIF-B12 10.8 0.504(c)


(a) Average value for IF-B12 is 0.463 0.01

(b) Included in the average minus two standard
deviations from IF-B12

(c) Included in the average plus two standard
deviations from IF-B12






69
Figure XIV depicts the elution profiles obtained when 20 pg of IF-B12

and NIF-B12 were chromatographed on Sephadex G-150. The profile for IF-B12

would indicate the presence of associated species while that for NIF-B12

would not. This is in direct correlation with the work reported by Allen

and Mehlman (9,10).

Determination of the equilibrium constants for binding of IF-B12 and

TIF-B12 complexes to the ileal receptor. The double reciprocal plots for

the equilibrium binding of IF-B12 and TIF-B12 to the guinea pig ileal homog-

enate are shown in Figure XV. Both sets of data obey a strictly linear

relationship and are essentially coincident. The equilibrium constant for

the binding of IF-B12 was found to be 5.5 x 109M-1 while that of TIF-B12

was 5.6 x 109M-1. The number of receptor sites available to each vitamin

B12 complex, indicative of the total amount bound at infinite concentra-

tion of vitamin B!2 complex, was found to be 1.4 x 1012 sites/gm wet

weight for IF-B12 and 1.1 x 1012 sites/gm wet weight for TIF-B12. The

IF-B12 complex and the TIF-B12 complex, therefore, bind equally avidly

to the guinea pig ileal receptor homogenate.

The kinetics of attachment of IF-B12 and TIF-B12 to the guinea pig ileal

receptor homogenate. The second order rate plots for the attachment of

IF-B12 and TIF-B12 to the guinea pig ileal receptor homogenate are shown

in Figure XVI. From the slopes of these lines, the second order rate con-

stants for the forward reactions (1.5 x 106M-1 sec-l for IF-B12 and

1.7 x 106M-1 sec-l for TIF-B12) can be calculated. These values are not

appreciably different.

Using these values and the equilibrium constants determined previous-

ly (5.5 x 109M-1 for IF-B12 and 5.6 x 109M-1 for TIF-B12), the rate con-

stants for the dissociation of the receptor IF-BI2 complex and the recep-















o- I 1x
0 0 X 40
I R V c e x
XX
X X

FII X X












A column of Sephadex G-150 (2.6 x 90 cm) was equilibrated at
O-











of 8 rs. at 37C. The complexes were dialyzed against the
100 200 300 400
EFFLUENT. ML.

FIGURE XIV molecular Sieve Chrotohromath Using r -150 Saphadex

of the Vitamin B12 Comoplxes of IF and NIF

A column of Sephadex G-l50 (2.6 x 90 cm) was equilibrated at

40C. using 0.0I K2HP04, 0.75M NaCi, pH 7.50. Both IF (20 pg)

and NIF (20 jig) were complexed in this same buffer with a 10-

fold excess of [57Co]cyanocobalamin (15 iCi/ig) for a period

of 8 hrs. at 370C'. The'complexes were dialyzad against the

equilibrating buffer and separately chromatographed at 25C.

Fractions (3.0 ml) were collected and the radioactivity in

each was counted. (x), IF-B12 complex; (o), NIF-B12 complex.














o

0



-,
\. I




S 2 3 4 5 6 7 8 (xlO! V1 )
I / EIF-BI2 free
12 free

FIGURE XV Double Reciorocal Plot for Determination of the Equilibriu.

Constant for the Binding of the IF-B12 Complex

and the TIF-BI2 Complex to the Guinea

Pig Ileal Homogenate at 250C.

Into albumin coated tubes (10 x 75 mm) was placed 0.74 ml KRPO4 buffer,

0.06 ml of the vitamin B12-protein complex in water, and 0.20 ml of the

guinea pig ileal homogenate. -Incubation took place for 180 min. in this

and in an identical solution containing KRPO4 buffer lacking calcium and

magnesium but .containing O.OOM Na2EDTA. The concentration of the vita-

min B12 complexes was varied from 1.0pg (as B12) to 450 pg (as B12).

The reactions were terminated by filtration on 1.2 micron Millipore fil-

ters coated with albumin and, following washing, the radioactivity on

each was counted. The Na2EDTA inhibitable counts were used to construct

these plots. (x), IF-B12 complex; (o), TIF-B12 complex.







S72
O
0 _________20__60_600_840__080
0

0
'I
r (

Co- x ----
I (D X-- -- .X-- --X- -- X"

U

o


120 360 600 840 1080
TIME., SEC.

FIGURE XVI Kinetics of Attachment of the IF-B12 Complex and the TIF-B12 -

Complex to the Guinea Pig Ileal Homonofnate at 250C.

For each protein-vitamin B12 complex, a total of 10 guinea pig ileal homog-

enate assay incubations were prepared using 0.2 ml guinea pig ileal homog-

enate and enough of each protein-vitamin B12 complex to equal 60 pg vita-

min B12. Incubation was conducted for a period from 2 to 20 min. and

the reactions were quenched by rapid filtration on 1.2 micron Millipore

filters soaked in albumin. The radioactivity on each was counted and the

Na2EDTA inhibitable binding was used to construct these second order rate

plots. Each point represents an average of two determinations. (o),

IF-B12 complex; (x), TIF-B12 complex.






73
tor TIF-B12 complex can be calculated. For IF-B12, this calculated rate

constant is 2.7 x 10-4 sec-1, while for TIF-B12, the value is 3.0 x 10-4

sec-1. These rate constants are also not appreciably different and demon-

strate that trypsin treatment of IF followed by complexation with vitamin

B12 does not grossly alter its interaction with the ileal receptor

homogenate.

Assessment of the vitamin B12 binding activity of neuraminidase treated

IF and TIF. IF and TIF were incubated with neuraminidase in a constantly

dialyzed system. Control incubations were conducted under the same con-

ditions without neuraminidase. Both enzyme treated and control samples

had vitamin B12 binding activity and immunoreactivity (Table 12). It is

clear, that although the vitamin B12 binding activity decreased markedly,

it does so to the same extent in the control preparations. The reason

for this is not fully clear, but when compared to the results obtained

using the BSA coated tubes, it seems that IF may have been physically

adsorbed to the containers. Attempts to stabilize IF in the presence of

sialinized glass tubes were not successful. The recovered material was

still uniformly sensitive to anti-IF antibody (Table 12).

In the experiment where IF and TIF were treated with neuraminidase

.in albumin coated tubes, the vitamin B12 binding activity remained about

90% of that originally present (Table 13). Again, there is no difference

between the experimental and control values. Likewise, this same table

shows that all the preparations are still sensitive to anti-IF antibody.

It is apparent that neuraminidase treatment did not alter the quantity

of vitamin B12 bound nor did it alter sensitivity to anti-IF antibody.

Polyacrylamide disc gel electrophoresis. The vitamin B12 complexes of

all four preparations (IF, NIF, neuraminidase treated IF and neuraminidase






74
TABLE 12

The Effect of Neuraminidase Treatment on the Vitamin B12 Binding Proteins


Charcoal binding assay

Antibody inhibition assay


Material Vitamin B12 Binding(a)(ng) Antibody Sensitivity(b)(%)

Initial Final Initial Final

IF 11 2.0 99 92

TIF 11 3.2 99 92

IF-Neuraminidase
treated 11 4.4 99 98

TIF-Neuraminidase
treated 11 6.2 99 99





75

TABLE 13

The Effect of Neuraminidase Treatment on the Vitamin B12

Binding Proteins Stabilized by Bovine Serum Albumin


Charcoal binding assay

Antibody inhibition assay


Material Vitamin B12 Binding(a)(ng) Antibody Sensitivity(b)(%)
Initial Final Initial Final

IF 58 49 99 97

TIF 51 46 99 99

IF-Neuraminidase
treated 57 49 99 98

TIF-Neuraminidase
treated 43 40 99 99





76
treated NIF) were subjected to polyacrylamide disc gel electrophoresis.

Table 14 summarizes the Rf values (relative to Bromphenol Blue) obtained

for these proteins incubated either in the presence or absence of BSA.

The Rf values for any single protein are identical in both systems. This

indicates that, although BSA coated the glass tubes in the second experi-

ment, it did not significantly alter the mobility of the vitamin B12

binding proteins on gel electrophoresis. The finding of interest is that

the neuraminidase treated proteins, IF and TIF, have the same Rf value,

but this value is significantly different (0.01 level) from the untreated

IF and TIF. The profiles of these gels are presented in Figure XVII for

the dialyzed experiment and in Figure XVIII for the albumin stabilized

materials. It is clear that neuraminidase treatment alters the electro-

phoretic mobility of both IF and TIF in an identical fashion, presumably

by altering the net charge of the molecules by removal of sialic acid.

Since this preparation of neuraminidase contained protease activity, it

may be that this change is secondary to proteolytic activity.

Binding of the neuraminidase treated IF and TIF fractions to the guinea

pig ileal homogenate. The vitamin B12 complexes of IF, TIF, and their

neuraminidase treated counterparts, from both experiments, were assayed

for their ability to bind to the guinea pig ileal receptor using the

standard guinea pig ileal homogenate binding assay (Appendix X). The

data are presented in Figure XIX for the albumin stabilized fractions.

It is clear that the neuraminidase treated materials bind to the receptor

just as avidly (Keq = 4.0 x 109M-1), and have as many potential binding

sites (1.7 x 1012 sites/gm wet weight) as their untreated controls. It

is evident that neuraminidase treatment of neither IF nor TIF destroys

its ability to bind to the guinea pig ileal homogenate.






77
TABLE 14

Polyacrylamide Disc Gel Electrophoresis of the Vitamin B12

Complexes of Intrinsic Factor and Trypsin Treated Intrinsic

Factor and Their Neuraminidase Treated Analogs(a)


Material Rf Without Albumin(b) Rf With Albumin(c)

IF 0.42 0.41

TIF 0.42 0.40

IF-Neuraminidase
treated 0.37 0.37

TIF-Neuraminidase
treated 0.37 0.38


(a) Rf values calculated relative to migration of Bromphenol Blue
(b) Neuraminidase treated while dialyzed

(c) Neuraminidase treated in albumin coated glassware



















0
0





C ..X
x0.x
40



0o x
N o-o..o--o---ci'0 o',X


0.1 0.2 0.3 0.4 05 0.6 0.7 0.8 09
MOBILITY RELATIVE TO BROMPHENOL BLUE

FIGURE XVII Polyacrylamide Disc Gel Electroohoresis of the

IF-B12 Complex and Its Neuraminidase Treated Analog

The vitamin B12 complexes of IF and neuraminidase treated IF

were prepared by dialysis against 1000 volumes of distilled

water containing a five-fold excess of [57Co]cyanocobalamin

(100. Ci/pg). The complexes were subjected to polyacrylamide

disc gel electrophoresis on the standard 7.5% polyacrylamide

gel system described in Appendix IV. (x), IF-B12 complex;

(o), neuraminidase treated IF-B12 complex.


~_ ~ IU~U I~---r--IC-CIC~----lll~-ll~-LI



















8 I
o x

Sx a xl
o .


0.1 02 0.3 0.4 0.5 0.6 0.7 0.8 0.9
MOBILITY RELATIVE TO BROM2PHENOL BLUE
FIGURE XVIII Polvacrylamide Disc Gel El.ctrorhorcsis of the
TIF-B12 Complex and Its 'euraninidase Treated Analog

The vitamin B12 conmlexes of TIF and neuraminidase treated TIF
were prepared by dialysis against 1000 volumes of distilled wa-
ter containing a five-fold excess of [57Co]cyanocobalamin (100

pCi/pg). The complexes were subjected to polyacrylamide disc
gel electrophoresis on the standard 7.5% polyacrylamide gel

system described in Appendix IV. (x), TIF-B12 complex; (o),
neuraminidase treated TIF-B12 complex.









o0
o

c 0 C O
ri
(11


0J










FIGURE XIX Double Reciorocal Plot for the Determination of the Eauilibrium

Constant for Binding of the Trypsin and -lu,-urainidase Treated

IF-B12 Complexes to the Guinea Pig Ileal Homogenate at 250C

Into albumin coated tubes (10 x 75 mm) was placed 0.74 ml KRPO4 buffer,
0.06 ml of the vitamin B12-protein complex in water, and 0.20 ml of the


guinea pig ileal homo-enate. Incubation took place for 180 min. in this
and an identical solution containing KRPO4 buffer lacking calcium and mag-

nesium but containing 0.0011_ Na2EDTA. The concentration of the vitamin B12
CM y

























complexes was varied from 1.0 pg (as B12) to 450 pg (as BM2). The reactions


were quenched by filtration on 1.2 micron Millipore filters coated with al-
bumin and the radioactivity on each was counted. The Na2EDTA inhibitable
counts were used to construct these plots. (o), IF-B12 coda plex; (4),

TIF-B12 complex; (e), neuraminidase treated IF-B12 complex; (A), neuramin-

ida.se treated TIF- n B12 complex.






81

The binding of IF-B12 to the neuraminidase treated receptor. The equilib-

rium constants and the number of binding sites available for IF-B12

binding to control and neuraminidase treated receptor homogenates were

graphically derived from the double reciprocal plot shown Figure XX. The

control and neuraminidase treated materials do not have different equilib-

rium constants for binding of IF-B12 (2 x 109M-I), nor do they differ

in the number of binding sites available (7.5 x 1012 sites/gm wet weight).

Furthermore, these Keq values are not appreciably different from those

derived using fresh guinea pig ileal homogenates. It, therefore, seems

clear, that neuraminidase treatment of the ileal homogenate does not

affect its binding of the IF-B12 complex.

Figure XXI shows the data for binding of IF-B12 to the neuraminidase

treated guinea pig ileal homogenate. Also included in this figure, are

points for the binding of TIF-B12 and both IF and TIF treated with neura-

minidase and completed to vitamin B12. It is evident that all the data

points lie very close to the line established by IF-B12 binding. There-

fore, these data indicate that all four of these preparations bind to the

neuraminidase treated guinea pig ileal homogenate to about the same extent.



The Effect of Trypsin and Neuraminidase Treatment of Intrinsic

Factor on the Transport of Vitamin B12 Across the Ileal Cell

Measurement of the transport of vitamin B12 across the ileal cell. The

fractional amount of vitamin B12 transported to the interior of each

everted ileal sac is defined as the ng vitamin B12 inside the sac per mg

tissue divided by the total ng vitamin B12 present at the initiation of

the experiment. The results for each vitamin B12 binding protein were

an average of three sacs excluding those that were visibly deflated. The










0x


0o 0
-Q C\

ai I




V)



2 4 6 8 10 12(xIO10M-1
I /CIF-82 fee
12 free

FIGURE XX Double Reciorocal Plot for the Determination of the Equilibrium

Constant for Binding of the IF-B12 Complex to the Guinea Pig
leal Honoeenite and to the '1eur:riinidase Treated Homonenate

at 250C

Into albumin coated tubes (10 x 75 mm) was .placed 0.74 ml KRPO4 buffer,

0.06 ml of the IF-B12 complex in water, and 0.20 ml of either the guinea

pig ileal homogenate or the neuraminidase treated honogenate. Incubation
took place for 180 min. in these and in identical solutions containing
KRPO4 buffer lacking calcium and magnesium but containing 0.001M Na2EDTA.

The concentration of the IF-B12 complex was varied from 1.0 pg (as B12) to
450 pg (as B12). The reactions were quenched by filtration on 1.2 micron

Millipore filters coated with albumin and the radioactivity on each was

counted. The Na2EDTA inhibitable counts were used to construct these plots.

(o), acetate treated control; (x), neuraminidase treated homogenate.






















5 15 25 35(xiO1 M-l)
I /EIF-Bi23free

FIGURE XXI Double Reciprocal Plot for Determination of the Equilibrium
Constant for Binding of the IF-B12, TIF-B1?, IFN-B,2 and TIFN-B12
Complexes to the Neuraminidase Treated Ileal Homogenate at 25C
Into albumin coated tubes (10 x 75 mm) was placed 0.74 ml KRPO4 buffer,
0.06 ml vitamin B12-protein complex in water, and 0.20 ml neuraminidase
treated homogenate. Incubation took place for 180 min. in these and i-
dentical solutions containing KRPO4 buffer lacking calcium and magnesium
but containing 0.001lh Na2EDTA. The concentration of the complexes varied
from 1.0 pg (as B12) to 450 pg (as B12). Reactions were quenched by fil-
tration on 1.2 micron Millipore filters coated with albumin and the radio-
activity on each was counted. The Na2EDTA inhibitable counts were used to
construct these plots. (o), IF-B12; (o), TIF-B12; (x), IFN-B12; (z),

TIFN-B12
12






84

results for each vitamin B12 binding protein were then divided by the

average value for NIF-B12 so that a relative amount of transport could

be defined in each case. These results are presented in Figure XXII.

As can be seen, relative to NIF-B12, all four proteins tested (IF, TIF

and their neuraminidase treated analogs) increase the amount of [57Co]-

cyanocobalamin appearing inside the gut sacs by approximately 8 fold.

There is no statistically significant difference between any of these

four preparations. It is clear, that within the limitations of this

experiment, treatment of IF with either trypsin or neuraminidase or with

trypsin followed by neuraminidase has no gross alteration on its ability

to effect the transport of vitamin B12 across the ileal cell.











0--
-- -- -







NF-2 F- TIF- IFN-B TIFN-
FIGURE XXII Effect of Trypsin and nidase Treatment of IF







on the Transport of Vitamin B? Across the Ileal Cell at 370C
Ileal gut sacs were prepared and everted. Into the interior of

each sac was placed 0.5 ml 0.14M NaC1, 0.005M KC1, 0.000T7 K2HPO4,

0.0056:1 glucose, 0.025M1 Tris HC1, 0.005M CaC12, pH 7.10, containing
50,000 units penicillin per liter. The sacs were suspended for 2 hrs.
in 7.0 ml of this buffer containing 1.0 ng vitamin B12 complexed to

each protein. The sacs were rinsed 3 times, opened, and the fluid
inside was removed and counted for radioactivity. Transport was de-

fined as ng vitamin B12 inside / mg tissue, divided by total ng vita-
Ld



NiF-B^ IF-B^ TIFPB IFN-B^ TIFN-B^










min GURE2 added. The average value for each protein was divided by
the average for NIF- Vi2 to yield relative transport. at 37
Peal gut sacs were prepared and everted. Into the interior of
each sac was placed 0.5 ml 0.14M NaCl, 0.005!. KC1, 0.0007/ K2HP04,

0.0056:1 glucose, 0.025M Tris HC1, 0.005M CaC12, pH 7.10, containing
50,000 units penicillin per liter. The sacs were suspended for 2 hrs.

in 7.0 ml of this buffer containing 1.0 ng vitamin B12 complexed to
each protein.- The sacs were rinsed 3 times, opened, and the fluid

inside was removed and counted for radioactivity. Transport was de-
fined as ng vitamin B12 inside / mg tissue, divided by total ng vita-
min B12 added., The average value for each protein was divided by

the average for NIF-Bl? to yield relative transport.












DISCUSSION


Purification of IF and NIF

The two classical vitamin B12 binding proteins, IF and NIF, from

hog pylorus, have been purified to homogeneity as evidenced by single

bands on both polyacrylamide and SDS polyacrylamide gel electrophoresis.

Furthermore, the far more difficult task of separating IF from NIF has

been accomplished in a facile one-step procedure. Allen and Mehlman

(9,10) were able to accomplish such a separation using a vitamin B12

ligand which lacked the 5,6-dimethyl-benzimidazole ring. This ligand,

produced by vigorous acid digestion, was coupled to Sepharose. Under

exacting LO:,Jitions of flow rate, te:ipercdture, and protein to ligO-nJ

ratio, good separations were obtained in 1.OM guanidine HC1. They attri-

bute the success of this column to the differential binding of IF versus

NIF to this ligand. In our hands, their results with this procedure

could not be duplicated.

Our procedure, however, employs only one step with high recoveries

of IF. It also requires only one column synthesis. More importantly,

it is effective at two flow rates (15 ml/hr.and 50 ml/hr.) and at two pro-

tein to ligand ratios (1.1 mg/jmole 812 and 105 mg/pg B12). Theoreti-

cally these results indicate that IF can be separated from NIF not by

virtue of the binding constant to vitamin B12 (both of which are the

same), but by the concentration of guanidine HC1 required differentially

to denature each molecule.





87

The work using hydroxyl apatite chromatography has also allowed

the separation of a vitamin B12 binding material we refer to as Fx4.

The nature of this vitamin B12 binder and its physiological role are

currently unknown.

The purified vitamin B12 binders used in these studies had specif-

ic activities of 9.5 ig vitamin B12 bound/mg protein for NIF and 11.2

ug vitamin B12 bound/mg protein for IF as determined by the modified

charcoal binding assay of Gottlieb. However, when the IF preparation

was incubated for 12 hrs. in an albumin coated tube containing equimolar

amounts of vitamin B12, and then passed over a G-25 Sephadex column

equilibrated with 0.01M K2HP04, 0.15M NaCl, pH 7.4, containing 100 ng

vitamin B12/ml, a peak of radioactive protein appeared in the column

void volume which had a specific activity of 29.8 ig vitamin B12 bound/

mg protein. Similarly, NIF, equilibrated in the same fashion, has a

specific activity of 17 ig vitamin B12 bound/mg protein. These data

argue that the charcoal binding assay is falsely depressed with highly

purified vitamin B12 binding proteins which are readily adsorbed to

glass. This depression of the charcoal binding assay can be corrected

by allowing all tubes and glassware utilized in the assay to stand for

2 hrs. in a 1 mg/ml BSA solution. After aspiration of the BSA solution,

use of this glassware to measure the specific activity of the vitamin B12

binding proteins gives values of 30 pg vitamin B12 bound/mg protein for

IF and 17.5 yg vitamin B12 bound/mg protein for NIF. These results indi-

cate that IF adsorption to glass causes artifacts in vitamin B12 binding

measurements made with the charcoal binding assay.