Title: Lipoprotein structure
Full Citation
Permanent Link: http://ufdc.ufl.edu/UF00099135/00001
 Material Information
Title: Lipoprotein structure apoprotein interactions in human plasma high density lipoproteins
Physical Description: xi, 137 leaves : ill. ; 28 cm.
Language: English
Creator: Grow, Thomas Ellis, 1945-
Publication Date: 1977
Copyright Date: 1977
Subject: Blood lipoproteins   ( lcsh )
Biochemistry and Molecular Biology thesis Ph. D
Dissertations, Academic -- Biochemistry and Molecular Biology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
Thesis: Thesis--University of Florida.
Bibliography: Bibliography: leaves 132-136.
Statement of Responsibility: by Thomas Ellis Grow.
General Note: Typescript.
General Note: Vita.
 Record Information
Bibliographic ID: UF00099135
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000074407
oclc - 04673926
notis - AAH9681


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I dedicate this dissertation to my best friend William

A. Patterson and the Krenu Construction Company.


The author would like to express his deep appreciation to

Professor Melvin Fried, his research director, for giving him guidance

and freedom of thought and action during the completion of this work.

The environment of Professor Fried's laboratory was exactly what the

author required for productive work.

The author also wishes to thank his wife, Susan, for her patience,

understanding, and sacrifices made during the last four years.

Special thanks are given to the author's advisory committee and

members of the faculty of the Department of Biochemistry for their

suggestions and criticisms during the execution of this research.

The author also thanks his parents for their understanding during

his years of education.


ACKNOWLEDGEMENTS ...............

. . i

LIST OF TABLES . . . . . . . . . . . . .

LIST OF FIGURES . . . . . . . . . . . .


ABSTRACT . . . . . . . . . . . . . .

INTRODUCTION . . . . . . . . . . . . .

Plasma Lipoproteins ... ... .. .... .........

Apoprotein Structure and Function ..............

Lipoprotein Structure ....................

Alternative Classification of Lipoproteins ..........

Exchange Reactions of Lipoproteins .............

Experimental Rational ....................

Research Objectives ... ..................

MATERIALS AND METHODS .....................

Materials . . . . . . . . . . . . .

Methods . . . . . . . . . . . . . .

RESULTS AND DISCUSSION .....................

Isolation and Purity of Lipoprotein Preparations ......

Crosslinking of Total HDL (HDLT) with DFDNB .........

Effect of Crosslinking on HDL Structure ...........

Crosslinking of HDL2 and HDL3 with DFDNB ..........

Crosslinking of HDLT with DMS and DFDNDPS ..........

Temperature Dependence of Crosslinking. ...........























Interpretation of Crosslinking Results. ... .. .. .. .59

Apoprotein Exchange Between HDL2 and HDL3 . .. .. ... .. .61

Effect of Crosslinking and Monofunctional Labeling
on Apoprotein Exchange. .. .. .. .. .. .. .. ... .66

Effect of Temperature and Dilution on Peptide Exchange .. .69

Rate of Peptide Exchange ... .. ... .. .. .. .. .69

Effect of Bromide lan on Protein Exchange .. .. .. .. .. .80

SDS-Polyacrylamide Gel Analysis of Exchanged Proteins .. .. .80

Interpretations of Protein Exchange Experiments .. . ... .85

Lipid Exchange Between HDL2 and HDL3 in Solution .. .. .. .88

Binding of Labeled HDL to Sepharose .. .. . .. .89

Peptide Exchange Using Sepharose-Bound HDL .. .. .. .. .91

Ultracentrifugal Analysis of Peptide Exchange Using
Sepharose-Bound HDL . .. ... . .. . . . . 91

SDS-PAGE Analysis of Exchanged Peptides Using
Sepharose-Bound RDL . .. .. .. .. ... . 96

Incorporation of Labeled Lipid into RDL2 and HDL3 ... .. 103

Lipid Exchange Between HDL3 and Sepharose-HDLj .. .. .. 106

Effect of Temperature and Dilution on Lipid Exchange .. .. 112

Effect of Saturating Cold Lipid on Labeled Lipid Exchange .. 112

Effect of Crosslinking on Lipid Exchange .. .. .. . 118

Effect of Added Organic Solvent on Exchange of Lipids
and Proteins . .. .. .. .. . .. . .. . 118

Interpretations of Sepharose-HDL Peptide and Lipid
Exchange Experiments ... .. .. .. .. . . . 125

CONCLUSIONS AND SPECULATIONS . ... .. .. ... . .. 128

BIBLIOGRAPHY .. .. .. .. .. .. . .. . . . 32

BIOGRAPHICAL SKETCH .... .. .. ... .. .. . .. 137










Bifunctional Reagents ...........

Amino Acid Analysis of 46,000 Dalton
Crosslinked Product from RDLT Cross-
linked with DFDNB... .........

Effect of Amount of CNBr Activation
on the Amount of Binding of HDL3 by
Sepharose 48 ........

Peptide Exchange Using Sepharose-Bound HDL

Removal of Labeled Lipid from Sepharose-
Bound HDL3 by SDS Wash ..........

Effect of Temperature and Dilution on
Lipid Exchange Between S-HDLg and RDL3 ..

Effect of Saturating Cold Lipid on Lipid
Exchange . . . . . . . . .

Effect of Crosslinking on Lipid Exchange .

. 90

. 92

. . .107

. . ..117

. .. .119

. ...120

. . .68

. . .71

. . .73

. . .75




1 SDS-PAGE of solubilized apoproteins
of IlDLy . . . . . .

2 Separation of HDL2 and HDL3 A d=1.125gm/cm3. ..

3 SDS-PAGE of RDLT crosslinked with DFDNB ......

4 Experimental approach to amino acid analysis
of crosslinked products .............

5 Comparison of the SDS-PAGE methods of
Fairbanks and Lammli for the separation





of highly crosslinked RDLT ............

,S-PAGE of HDL2 crosslinked with DFDNB .......

,S-PAGE of HDL3 crosslinked with DFDNB .......

IS-PAGE of HDLT crosslinked with DMS ........

IS-PAGE of HDLT crosslinked with DMS (5% BME). ...

IS-PAGE of HDLT crosslinked with DFDNDPS ......

S-PAGE of HDLT crosslinked with DFDNDPS
(5% BME). . . . . . . . . . . .

change of apoproteins between [12I]-HDL3
and HDL2 . . . . . . . . . .
change of apoproteins between [ I]-HDL2

. . .










6 SD

7 SD

8 SD

9 SD

10 SD

11 SD

12 Ex

13 Ex

d na HD

14 Effect of crosslinking on apoprotein exchange .

15 Effect of FDNB labeling on apoprotein exchange

16 Effect of crosslinking the unlabeled subclass
on apoprotein exchange ...........

17 Effect of temperature on apoprotein exchange .

18 Effect of temperature and dilution on
apoprotein exchange .. . .. .. . .77

19 Trime course of upoprotein exchanglpe .. .. .. .79

20 Effect of KBr on apoprotein exchange .. .. .. .82

21 Polyacrylamide gel analysis of exchanged apoprotein .84

22 Polyacrylamide gel analysis of exchanged apo-
proteins: Effect of crosslinking with DFDNB. .. .87
23 Suunitexchnge etwen S-125I]HL n D 9
24 Submnit exchange between S-[12I]-HDL2 and HDL2 9

25 Polyacrylamide gel analysis of available
apoproteins from S-HDL .. .. .. .. .. .. 100

26 Polyacrylamide gel analysis of apoproteins
exchanged from S-[125I]-HD) L ..... 102

27 Uptake of labeled lipids by HDL3 .. .. .. .. 105

28 Effect of increasing amounts of cold HDL, on
lipid exchange .. .. .. .. .. .. 109

29 Effect of increasing amounts of cold HDL3 on
lipid exchange .......... 111

30 Rate of lipid exchange between S-HDL3 and HDL .. 114

31 Time course for lipid exchange between
S-HD~g and HDL3 .. .. .. ... .. .. .. 116

32 Effect of ethanol of the exchange of lipids
from S-HDL~ to HDL ........ 122

33 Effect of ethanol on the exchange of lipid
and protein from S-HDL~ to RDL3 .. ... .. .' 124

34 Model for HDL3 HDL2 interconversion .. . ... 131


Ai angstrom

Apo apoprotein

CD circular dichroism spectroscopy

Ci curie

dpm disintegrations per min.

IR infrared spectroscopy

LCAT Lecithin cholesterol acyl transferase

leu leucine

MW molecular weight

NMR nuclear magnetic resonance

ORD Optical rotory dispersion

val valine

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



Thomas Ellis Grow

June, 1977

Chairman: Melvin Fried
Major Department: Biochemistry

The modes of association of the major apoprotein -species of human

plasma high density lipoprotein (HDL) was investigated using bifunctional

crosslinking reagents. How these associations help to govern the struc-

ture of the RD)L subclass, HDL2 and HDL was studied by observing both

apoprotein and lipid exchange between HDL particles.

Crosslinking of HDL with a 5 to 10-fold molar excess of difluoro-

dinitrobenzene resulted in the production of high molecular weight

crosslinked products with the major product shown to be the combination

of the two major apoprotein species ApoA-1 and ApoA-2. The composition

of this product was confirmed by amino acid analysis.

Crosslinking with other bifunctional reagents indicatesspecificity

of initial reaction sites as well as some freedom of movement of the

apoproteins within the particle. However, the crosslinking patterns

obtained with the RDL subclasses were identical, suggesting specific

structural properties of HDL2 and HDL3

Apoproteins, labeled with 12i, were shown to exchange between and

among the RIDL subclasses. This bidirectional exchange process was

inhibited by crosslinking with bifunctional reagents and appeared

to depend upon collision complex formation. By the utilization of

Sepharose bound IIDL it could be shown that both free apoprotein mole-

cules, as well as subunits consisting of lipid-apoprotein combinations,

could exchange between HDL2 and HDL3. The exchange appears to involve

a conformational change in the apoproteins during the transfer process.

Radioactive cholesterol, cholesterol palmitate, and phosphatidy1

choline, bound to HDL after adsorption to celite particles, could be

shown to undergo rapid exchange between MDL particles. This exchange

also appears to depend on collision complex formation but was not

inhibited by chemical crosslinking of the apoproteins.

The rate of these exchange processes is significant in the life-

time of the lipoprotein particles in vivo: 2.5%/hour for apoprotein

exchange and >50%/hour for lipid exchange.

It was concluded that ApoA-1 and ApoA-2 must lie very close to one

another in the intact lipoprotein particle. The reproducibility of

the crosslinking experiments on HDL obtained from several donors suggests

that in the intact HDL particle the apoprotein components may be found

in a rather fixed spatial orientation to one another. It is also

suggested that there is a dynamic relationship between HDL2 and HDL .

Even though these two subclasses can exist as stable separate entities,

when they are present together in solution, significant interaction

between particles may occur. Exchange of lipids and apoproteins occurs

between KDL2-HDL2 and HDL -HDL as well as between HDL2 and HDL3 mole-


It is suggested that the interconversion of RDL2 and HDL3 may be

determined by the availability of certain lipids.


Plasma Lipoproteins

Plasma lipoproteins play an important role in mammalian energy

metabolism and also serve as the major transport particles for many

physiologically important molecular species. It has been suggested

that they are causally related to atherosclerosis since they are the

carriers for those substances deposited in the atheromatous plaque.

Circulatory system pathology found in hyperlipoproteinemics may result

from quite small alterations in lipoprotein structure; differences that

may be due to altered protein-protein or protein-lipid interaction.

Human serum contains four major types of lipoproteins: chylo-

microns (CHYL), very low density lipoproteins (VLDL), low density

lipoproteins (LDL), and high density lipoproteins (HDL). Although

these four classes appear to-differ in both lipid and protein compo-

sition as well as function, there are distinct interrelationships

between the particles. The presence of one class directly influences

the other classes (1).

Chylomicrons, formed in the intestinal mucosa and released into

the blood from the major lymphatic duct, function as the carrier of

many lipid types. Lypolytic enzymes remove lipids from these particles~

as they circulate. VLDL and LDL appear to be closely related as LDL

may be the result of VLDL delipidation (2). LDL functions as the

major cholesterol carrier in blood. HDL can be divided into two

subgroups; HDL2 and RDL3. HDL may be important in cholesterol

deposition disease (3).

The isolation of lipoproteins from serum or plasma is relatively

simple and may be accomplished by flotation in the ultracentrifuge.

As the density (d) of serum lipoproteins ranges from approximately

0.95 gm/cm3 to 1.21 gm/cm3, centrifugation of serumo, the density of

which has been raised to 1.21 gm/cm3 by the addition of salt, will

result in the lipoproteins floating to the top of the centrifuge tube

while the other proteins sink (4). Each lipoprotein class is charac-

terized by its own density range for isolation: CHYL and VLDL < 1.006,

LDL 1.006 1.063, and HDL 1.063 1.21. By stepwise increases in

density (4), or by density gradient centrifugation (5), these lipo-

protein classes may be prepared in relatively pure form. Although

other methods such as molecular sieve chromotography (6) and chemical

precipitation (7) have been described, the ultracentrifugal floatation

method remains the most widely used.

The protein and lipid compositions of these lipoprotein classes

have been studied and the following have been reported (1,8) (all

percentages on a dry weight basis).


HDL, whose molecular weight ranges from 180,000 to 350,000,

consists of approximately 50% protein, 22% phospholipids, 3% free

cholesterol, 14% cholesterol esters, and 8% triglycerides. Of the

phospholipid portion, 70% is phosphatidylcholine 14% is spingomyelin.,

5% phosphatidy1 serine and phosphatidyl ethanolamine, and the remain-

der other minor phospholipids. The protein portion of the RDL particle

consists of two major polypeptides, ApoA-1 (ApoGln-I) and ApoA-2

(ApoGln-II). The amino acid sequences of both polypeptides are known

(9,10,11) and ApoA-2 is known to be a dimer of two identical subunits

connected by a single disulfide bridge at position 6 (10). Another

group of smaller proteins, designated ApoC or the C-peptides, comprises

approximately 5% of the total HDL protein. At least three C-peptides,

C-I, C-II, and C-III, are recognized in HDL (2). The protein of HDL

contains 3-4% covalently bound carbohydrate including glucosamine,

fructose, galactose, mannose, and sialic acid (8). In addition to the

C-peptides, KDL also may contain the "thin-line" protein and "arginine

rich" protein as minor components (1).


LDL particles are larger than HDL with molecular weights ranging

from 2 to 3 million. They also contain a larger percentage of lipid

than HDL (75-78%); consisting of 36-42% cholesterol esters, 21-23%

phospholipids, 8-9% free cholesterol, and 6-8% triglycerides. Other

lipids may be present in smaller amounts. The proteins make up 20-22%

of the particle. The major polypeptide is denoted apoprotein B (ApoB),

a large protein of molecular weight 225,000. There may also be very

small amounts of the C-poptides described for liDL. Again, 3-5% carbo-

hydrate is found covalently associated with the protein.


VLDL particles cover a wide range of densities and molecular

weights (3 128 x 106) with at least two maxima at densities of 0.980

and 0.958 gm/cm3. They contain 90% lipid with an average composition

of 50-60% triglycerides, 10-12% free cholesterol, 4-6% cholesterol

esters, and 18-20% phospholipids. Protein represents only 8-12% of

which 40% is apoprotein B and the remaining 60% is divided among the

three C-peptides described for HDL. A small portion of ApoA may also

be present and it has been demonstrated that different fractions may

contain different amounts of apoproteins (2).


Chyl contain mostly lipid with only 1-2% protein. These very

large particles have molecular weights of 5 4300 x 108. The protein

consists of all types of previously described apoproteins found in the

other lipoprotein classes. It has been shown that human lymph ch-ylo-

microns contain approximately 66% ApoC, 22% ApoB, and 12% ApoA. The

lipids are predominately triglycerides (80%) with only about 8%

phospholipids and 5% cholesterol (1,2).

It is important to note the overlapping protein composition in

all of these classes. It is presumed that the specific protein compo-

sition of each is important in determining its total lipid content and

the specific type of particle that is formed. These proteins should

play a very important role in determining the function of the particle

as well (1).

Apoprotein Structure and Function


ApoC-I has been sequenced (12) and is a single polypeptide chain

of 57 amino acid residues (MW 8000). ApoC-I is known to function in

lipid binding as well as LCAT activation (13). Lipoprotein lipase

also appears to be activated by ApoC-I (14).

ApoC-II is the largest of the C-peptides withi 100 amino acid

residues and a molecular weight of about 12,500 (15). It also binds

lipids and is a potent activator of lipoprotein lipase (16).

ApoC-III, a single polypeptide of 79 amino acid residues has also

been sequenced (17). Attached to threonine-74,ApoC-III contains an

oligosaccharide moiety consisting of galactose, galactosamine, and

sialic acid (17). Although no direct physiological role is yet known,

ApoC-III has been used often for the study of lipid-protein inter-

actions (18-21).


The characterization of ApoB has led to many conflicting statements

concerning the number and size of the polypeptide subunits. Due to

technical problems associated with isolation of a soluble apoprotein,

a variety of physical methods have indicated molecular weights ranging

from 8,000 to 275,000 (22). However,.most investigators support the

idea of a large (275,000) molecular weight peptide. These problems

have also hindered studies on specific functions of ApoB and as yet

no definite physiological role other than lipid binding has been dis-


ApoA is a term used to describe the two most extensively studied

apolipoproteins, ApoA-1 and ApoA-2. ApoA-1, a single polypeptide of

molecular weight 28,330, contains 245 amino acid residues and has been

sequenced (8,23,24). Although the binding of phospholipids by ApoA-1

has been studied by several investigators, conflicting results on lipid

binding in the absence of ApoA-2 have been reported (25,26,27). These

differences in the extent of lipid binding by ApoA-1 may be due to

self-association of the polypeptides that may mask lipid binding sites

(28). Self-association has been demonstrated by both ultracentrifugal

and chemical techniques (1),

ApoA-2 is a polypeptide dimer of two identical chains with a

molecular weight of 17,500 (29). Each chain contains 77 amino acid

residues. The d-isuffide bond occursu between thec single cysteine

residues that occur at position 6 (29). The disulfide linked dimer

appears to be characteristic of only man and chimpanzee; the dimer form

apparently is not crucial for ApoA-2's lipid carrying role (1). ApoA-2

does bind lipid strongly and may increase the binding capacity of

ApaA-1 as well (2).

Although lipid binding is the only role as yet assigned to ApoA-2,

ApoA-1 appears to have several functions other than lipid binding in

the HDL particle. ApoA-1.serves as an activator of LCAT (30), may be

an acceptor of cholesterol and cholesterol ester during VTLDL catabo-

lism (2), and appears to have some function in regulating the lipid

content of membranes (31,32).

Lipoprotein Structure

The study of lipoprotein structure has been approached utilizing

three basic types of techniques: 1) spectroscopic methods (IR, CD,

ORD, X-ray, neutron scattering, NMR, and fluorescence); 2) physical

techniques (Ultracentrifugation and electron microscopy); and 3) chem-

ical methods (chemical modification, amino acid analysis, bifunctional

reagents, reassembly). The actual composition of lipoprotein particles

has been fairly well described, as indicated above, and only in the

case of L~DL is there significant disagreement on Apoprotein size and

composition (1,8). However, when three-dimensional structure is

considered, little is known at present regarding specific protein-

protein or protein-lipid spatial relationships or interactions in

intact lipoprotein particles.

VLDL and Chyl have proved difficult to study due to the signifi-

cant turbidity of their solutions. This fact has limited the use of

spectroscopic methods and as a result' only the techniques of ultra-

centrifugation, electron microscopy, and ge1 filtration have been

used. This, coupled with the fact that these classes consist of a

spectrum of a~ wide variety of sizes with differing compositions, has

prevented all but the most basic of structural studies. Ultracentri-

fugation techniques indicate the following: Chyl MW 5-4300 x 108

daltons, diameter 1200-11000 A, hydrated density 0.93gm/cm VLDL-

MW 3-128 x 106 daltons, diameter 300-700 A, hydrated density 0.93

1.0gm/cm3 (1). The only structural determinations made thus far for

these particles are based on thec surface activity of the protein

components which seems to indicate thaot some portion of the proteins

are located on the surface of the particle (2), The information now

available has led to the suggestion of a lipid-core model for the

triglyceride richChyland VLDL particles. This model assumes that the

surface of the particle is occupied by phospholipids, cholesterol, and

proteins, while the central core consists of the less polar and more

hydrophobic triglycerides and cholesterol esters (33). The quantities

of protein, phiospholipid and cholesterol in these particles appear to

be sufficient to cover the surface of the particles as predicted (34).

LDL structure has been studied by many of the different techniques

mentioned earlier (2,8,35,36,37,38). Data from electron microscopy

and X-ray diffraction indicate a spherical particle with neither well

defined nor significant subunit structure (2). Information from

analytical ultracentrifugation, as well as X-ray and electron micro-

scopy, give the following information: MIW 2.3-2.7 x 106 daltons,

hydrated density 1.028-1.034gm/cm3, diameter 216-220 A. In distinction

to Chyl and VLDL, LDL is rather uniform in size and composition. The

protein component appears to have about 25% a-helix, 37% B-structure,

and 37% random coil as determined by CD (38). Although this structure

appears to be temperature dependant, a recent report indicates the CD

spectrum may be greatly complicated by the lipids present in the

particle (39). Both NMR and fluorescence studies indicate that the

protein, although near the surface of the LDL particle, is highly

associated with the lipid components of the molecule (36,37). It has

also been suggested that the core of LDL is mostly composed of hydro-

carbon chains while the outer layer of the particle is sparsely

occupied by proteins that emerge from the lipid core (35); a model

similar to the lipid-core model for VLDL. Other investigators,

utilizing electron microscopy and X-ray scattering, have suggested a

significant degree of subunit structure that includes many protein

subunits (20-60 with MW 8,000 or 27,000) arranged with icosahiedral

symmetry. The protein subunits give a symmetrical appearance with

the surface area occupied by both protein and phospholipid (40,41).

These subunit models do not take into consideration recent findings

that indicate the major protein component consists of only two poly-

peptides of molecular weight 250,000 or greater (42).

HDL has been studied most extensively, probably because it is

the easiest of the particles to work with. As noted above, its pro-

tein composition is well known and the major polypeptides have been

sequenced. RDL can be divided into two subclasses, HDL2 and HDL ,

which appear to differ mainly in their molecular weights (HDL2

184,000) (1).~ X-ray studies have indicated that RDL consists of two

regions of differing electron densities: an outer shell and an inner

core. The inner core has a radius of approximately 43 A in HDL2

(37 A in HDL3) while the outer shell radius is 14 A (11 A in HDL3).

The electron density of the outer shell indicates that is consists of

polar lipid headgroups and proteins (43). The size of a fully extended

phospholipid headgroup is about 11 A, a value that fits in well with

the X-ray data. Fluorescence techniques yield information on porcine

HDL3 consistent with a peripheral location of the protein component,

with the suggestion that as much as 80% of the protein is located at

or near the surface (37). This hypothesis is further supported by the

'finding of Scanu that 90% of the lysine residues of the apoprotein

components of human HDL is accessible to succinylating agents (2).

This would indicate that most of the protein is near the surface as

there are many lysines rather evenly distributed throughout the pro-

tein chains.

CD of the protein portions of HDL indicate 70% a-helix, 11%

B-structure, and 19% random coil (38). The removal or alteration of

11pid components greatly affects the structure of HDL apoproteins.

Delipidation lowers the helicity to 52%. Delipidation followed by

relipidation with egg phosphatidyl choline changes a-helicity to 64%

while thle addition of cholesterol further increases the helicity to

70% (38). These observations indicate strong lipid-protein interactions.

Two basic models of HDnL that are consistent with spectroscopic and

chemical data have been proposed. Assman,et al. and Assman and Brewer

(22,44) have suggested a model similar to the membrane structure pro-

posed by Singer and Nicolson (45) in which the HDL protein is depicted

as an "iceberg" floating in a "sea of lipid." This model takes into

account the fact that portions of the helical regions of the apoproteins

are two-sided; that is, one side of the helix is polar while the other

is non-polar (46). This amphipathic organization of the helix could

allow simultaneous interaction of the helix with lipid, protein, and

the aqueous medium. Whether or not the helices are oriented perpen-

dicular or parallel to the surface of the particle isthe_ essential

difference between this model of Assman,et al. and Assman and Brewer

(22,44) and that of Jackson, et al. (25). Jackson has suggested that

the long axes of the helical regions are oriented perpendicularly to

the fatty acy1 chains of the phospholipids. In this model, carbon

atoms 2-4 of the fatty acy1 chains could interact with the non-polar

side of the helix, allowing the hydrophyllic portions to interact with

the medium or other protein chains. There is evidence to suggest that

little electrostatic interaction occurs between phospholipid polar

headgroups and the charged amino acid residues of intact lipo-

proteins (22,47,48).

Little is known of the protein-protein interactions that may occur

in intact lipoproteins. The only studies reported have been carried

out on human HDL apoproteins in vitro. It has been shown that if one

mixes the RDL apoproteins A-1 and A-2 together in equimolar amounts,

they appear to associate into an aggregate of molecular weight

46,000 (28). This A-1;A-2 complex will bind less lipid than would

be expected, indicating that some of the lipid binding sites are

blocked by protein-protein -interaction. Other studies indicate that

in the absence of lipids and other apoproteins, both A-1 and A-2 will

self-associate (49-53). Although such studies help to understand the

types of interactions that may occur, only work done on the intact

particle can shed light upon the actual associations and interactions

that govern the final structure and function of the lipoproteins.

Recent evidence from Friedberg and Reynolds (54) suggests that

A-1 and A-2 always exist in a molar ratio of 2 A-1 polypeptides to

1 A-2 polypeptide dimer in human HDL. Their study also suggests that

this ratio of 2:1 is constant from individual to individual as well as

from subclass to subclass (HD~L2 to HDL3). This would indicate that

perhaps, for ApoA-1 and ApoA-2, a specific polypeptide composition is

found in HDL2 (4 A-1 + 2 A-2) as well as in HDL3 (2 A-1 + 1 A-2).

It is evident that much is yet unknown concerning the structure

of lipoproteins and that experimental evidence derived from studies

utilizing intact particles and techniques that allow precise definition

of spatial orientation is needed.

Alternative Classification of Lipoproteins

It should be noted at this point that another meth~od of classifi-

cation for plasma lipoproteins has been suggested by Kostner and

Alaupovic (55). Based on immunochemical evidence, they have suggested

that lipoproteins occur in separate families, the composition of which

is determined by the presence of specific apoproteins. The three


families they describe, LP-A, LP-B, and LP-C, represent particles

containing ApoA-1 and/or ApoA-2 (LP-A), ApoB (LP-B), and ApoC-I-III

(LP-C). The important difference between the two methods of classi-

ficatiohn is that, according~ to Kostner and Alaupovic, a physically

defined density class, such as HDL, may contain not only particles

with just ApoA-1 and ApoA-2 but also particles with only ApoB and

only ApoC. One should realize, however, that HDL isolated as a density

class will contain mostly LP-A and LP-C with a very small amount of

LP-B (2%). This LP-B is only found associated with the hDL2 density

subclass suggesting the possibility of contamination by remnant LDL

particles. Even the presence of particles containing only LP-C could

be an artifact resulting from the generation of incomplete or altered

particles during the extensive ultracentrifugation required for the

isolation of the HDL density class. In any case, the actual differ-

ence between HDL as a density class and LP-A as described by these

investigators would only be the presence of the small amount of C

peptides generally believed to be associated with the HDL molecule.

Although the possibility of lipoprotein families is a question that

merits consideration, the results of the experiments described herein

would apply to either model of the lipoprotein particles.

Exchange Reactions of Lipoproteins

Plasma lipoproteins are known to be a very dynamic population of

macromolecules. They are continuously being synthesized and degraded

at a high rate. The amount of any individual lipoprotein class present

at a given time is determined by the balance between synthesis and

catabolism. The individual components of lipoproteins, however, are

not degraded or synthesized at the same rate. This is due to the fact

that both lipid and protein components can be exchanged between indi-

vidual lipoprotein molecules as well as between different classes of

lipoproteins (1).

Lipid Exchange

Phospholipids appear to exchange between all classes of serum

lipoproteins at rates dependent upon their relative concentrations in

the individual classes (56,57). When labeled phospholipids are incor-

porated into an individual class of lipoproteins and this class is

allowed to interact with other lipoprotein classes in vivo or in vitro,

an exchange reaction with equilibrium, reached in 4 to 5 hours, is

observed (58). This exchange is independent of any protein exchange

and occurs at significantly different rates for different phospho-

lipid types. Lysalecithin exchanges most rapidly, followed by lecithin

and sphingomyelin (57,59). No significant differences are observed for

subclasses of an individual phospholipid class (59). This exchange can

be increased in vitra by the presence of a phospholipid exchange

protein (60).

Cholesterol is known to exchange rapidly between all classes of

human serulm lipoproteins (61). This rapid exchange occurs both in vivo

and in vitro wFth equilibrium attained after 2 to 6 hours~ (62,63).

Cholesterol ester exchange between plasma lipoproteins in vivo and

in vitra has been both suggested and disputed (62,64,65). There is

evidence that seems to indicate that cholesterol enters can be trans-

ferred from LDL to other lipoproteins in vitro (66).

The exchange of other lipid types has been reported for human as

well as other animal lipoproteins. Triglycerides exchange between

different classes of lipoproteins (67) as do unesterified fatty acids

(68) and a-tocopherol (69)..

Protein Exchange

Although the exchange of lipids does not appear to depend upon

the simultaneous exchange of protein, it has been shown that apoprotein

exchange does occur. Only ApoC of HDL and VLDL has been shown to

exchange in vitro and in vivo (70,71). This exchange is bidirectional

and not like the apparent transfer of protein from VLDL to LDL (2).

Mechanisms of Exchange

The movement of either lipid or protein from one lipoprotein class

to another can represent an exchange, a transfer, or the combination

of the two processes. It is evident that both processes depend upon

the lipoproteins existing in a dynamic state.

The in vitro exchange of lipids has been the most studied and the

process appears to be a physicochemical one (72). Exchange could occur

by at least two mechanisms. First, it has been suggested that exchange

occurs when individual lipid molecules escape the lipoprotein particle

and enter the aqueous medium. They are then picked up by other lipo-

proteins or membranes (73,74). This model for exchange lacks strong

evidence in its support but may be the mechanism for certain specific

lipid types (74).

An alternate mechanism involves the formation of collision.complexes

between lipoproteins (75). This would allow for diffusion of lipid

and/or protein molecules between the particles and at the same time

would not require the thermodynamically unfavorable dissociation of

hydrophobic molecules into an aqueous medium.-

As both mechanisms have been supported with experimental evidence,

it is important that each be considered as a possible explanation for

any exchange reaction. Experiments that will belp distinguish

between the two mechanisms would include the observation of the effect

of factors that influence collision rates between molecules, i.e.

temperature, dilution, etc.

Although only lipid transfer mechanisms have been postulated,

the same types of models should be considered when observing protein

transfer or exchange.

Experimental Rational

In studying either protein structure or protein-protein inter-

actions occurrences of natural crosslinking due to disulfide bridges,

peptide linkages between lysine E-NH2 and glutamic or asparatic acid

y or B-COOH, dr carbohydrate bridges have been valuable in under-

standing the final active structure that is being considered, e.g.,

insulin, ribonuclease, collagen. As such crosslinks are often not

found where one might like to find them, the introduction of stable

covalent bridges or crosslinks can aid in the study of protein

structure (See Ref. (76) for a recent review of bifunctional reagents).

Bifunctional reagents are capable of producing three types of cross-

linked products: intramolecularly crosslinked proteins, intermolec-

ularly crosslinked homopolymers (identical subunits), and inter-

molecularly crosslinked protein complexes nonidenticall subunits or

different proteins). Such reagents have been used in a variety of ways

to determine such things as the spatial geometry of membrane components

(77-81), the mapping of ribosome proteins (82), and the subunit

structure of oligomeric proteins (83).

There are two basic types of crosslinking reagents: those that

are cleavable and those that are not. The advantage of cleavable

crosslinkers is that one may isolate the crosslinked product, cleave

the newly formed bridges, and identify the proteins that were cross-

linked. This technique is particularly useful when studying the

crosslinking of multicomponent systems such as membranes.

Table 1 lists a selection of different types of crosslinkers and

gives certain specific characteristics. The selection of reagents

with differing lengths, degrees of hydrophobic character,or reactive

group specificity can yield products that differ because of the specific

structure or physical environment of the proteins that are crosslinked.

For example, in studying the human erythrocyte membrane, Moxley and

Tsai were able to produce different crosslinking patterns by varying

only the length of the bifunctional reagent used (81).

As all lipoprotein particles are thought to contain two or more

polypeptide chains and since the structure and function of these

particles may depend to a great extent upon specific protein composition

and/or protein-protein inlteractions, investigation wlthl btfunctiolnal

reagents could prove to be a useful method to define some of these


Because the function and characteristics of individual lipoprotein

classes differ, and these.differences ultimately must be related to

structure, studies dealing with the interactions and associations of

Table 1.

Bifunctional Crosslinking Reagents


N ztCI NiallI Lysine, Tyrosine

3,C-O--(C-(CH, I C-O-CH,




1. Imidoesters
(i.e. dimethyl

2. Dinitro Difluoro-


F N(De

5-6 A

9-10 A

Lysine, Tyrosine

Lysine, Tyrosine

SH, E-Amino
(High pH)

3. Difluoro Dinitro- F ~ F
Diphenyl sulfone ( >-0

4. N, N-Di (Bromoacetyl)

Pd + HOAc

5. Dimethyl Dithiobis

8 Mercapto-

NH c

HzC -C-2-(CH,);-5- -(cH,a- c- o-CH,


the characteristic apoproteins of a given lipoprotein class should

prove of value in understanding how these characteristics are deter-

mined by the structure.

KDL, the best characterized of the lipoproteins, was used in the

experiments described in this report. Its small size, stable structure,

ease of isolation and handling, as well as its variety of polypeptides

made it the model of choice. Although no definitive physiological

role has yet been assigned to HDL, certain other characteristics make

this an interesting model for study. C-peptides such as are found in

HDL are known activators of LCAT and lipoprotein lipase, and Apo-1 is

known to activate LCAT specific for certain substrates (1). Reynolds

and Simon (28) have reported altered lipid binding upon associations

of the apoproteins of HDL. Perhaps this interaction is a mechanism for

regulation of lipid binding. As the different lipoprotein classes

appear closely related and even share many of the same components, both

lipid and protein, the actual association of the different protein

components may play a very important role in regulating both structure

and function for the different lipoprotein particles.

Research Objectives

The purpose of this research was to gain insight into the structure

of intact serum lipoprotein particles specifically, the high density

lipoprotein particle. This was approached by attempting to understand

the interactions and associations of the individual apoprotein compo-

nents of these macromolecules. To aid in organizing the information

gathered, the following objectives were set forth:

1. To determine if bifunctional reagents are capable of inducing

crosslinks between individual apoproteins in intact HDL

2. To characterize and identify any crosslinked products pro-


3. To evaluate the result of the use of crosslinking reagents of

differing length and hydrophobic or hydrophyllic character

4. To determine and explain any possible differences in the

crosslinking patterns produced from HDL2 vs. RDL3

5. To discover if the apoproteins and lipids of intact HDL

exchange between the separate HDL subclasses

6. To investigate any possible effects of chemical crosslinking

on such exchange processes

7. To gain a greater understanding of the relationships and

structures of the two HDL subclasses, HDL2 and HDL .




1,5-difloro, 2,4-dinitrobenzene (DFDNB) and sodium dodecyl sulfate

(SDS) were purchased from Sigma Chemical Co., St. Louis, Missouri.

4,4'-difluoro 3,3' -dinitradiphenylsulfone (DFDNPS); dimethyl suber-

imidate dihydrochlorida (DMA); and dimethyl 3,3'-dithiobispropionim-

idate dihydrocholoride (DTBP) were purchased from Pierce, Rockford,

Illinois. Reagents for acrylamide gel electrophoresis were obtained

from BioRad Laboratories, Los Angeles, California. All other chemicals

used were reagent grade and purchased from common suppliers.

Radioisotopes and Scintillation Materials

[12IJ-Nal; [choline-methyl-14C]-phosphatidy1 choline, specific

activity 50mCi/mmole; [11-12 3H]-cholesterol, specific activity

60Ci/mmole; [11-12 3H]-cholesterol palmitate, specific activity 294mCi/

mmole; Aquasol-2; and mini-vials were all purchased from New England

Nuclear, Boston, Massachusetts.


Whole human blood was obtained fresh from healthy male donors or

as outdated blood units from the Civitan Regional Blood Center, Gaines-

ville, Florida. In no instances were the results of any repeated

experiments different depending upon the source of blood.


Isolation of Lipoproteins

Whole blood was centrifuged for 20 min at 2000 RP:M using an IEC

Model SBV centrifuge to remove red cells. The clear serum or plasma

was removed by decanting and the density then raised to 1.063gm/cm3

by the addition of solid KBr. This solution was centrifuged for

20-24 hrs.at 42,000 using a Beckman Ti60 rotor in a Beckman model L2-65B

ultracentrifuge. After centrifugation, the top 3-5 ml containing

CHYL, VLDL, and LDL were removed by aspiration and discarded. The

remaining solution was adjusted to density = 1.21gm/cm3 by the addition

of more solid KBr. The serum was again centrifuged for 24 hrs. at

42,000 RPM as above. The top 2-3 ml of each tube were then removed

using a Pasteur pipet and pooled. When total HDL (HDLt) was desired,

the pooled lipoprotein solution was further purified by another centri-

fugation step. This was carried out as above after the crude HDL

solution was adjusted to density = 1.063gm/cm3 by the addition of 0.15M

NaC1 containing 10 ME EDTA and 0.02% NaN3. Each tube was underlayered

with 5-10 ml of KBr solution of density = 1.21gm/cm~ before centri-

fugation. After centrifugation, the top 5 ml of each tube (LDL

contamination) was removed by aspiration and discarded. The yellow

band in the tube located above the 1.21 density layer was removed using

a Pasteur pipe and saved as purified total RDL. This HDL solution was

immediately dialyzed against 2 x 4 liters of 0.15M NaCl containing

10 ME EDTA and 0.02% NaN3, and stored at 4'C until use.

When the separate RDL subclasses, HDL2 and HDL were desired, the

following procedure was used. After the three ultracentrifugation

steps described above, the total HDL was dialyzed against a Kllr-NaC1

solution of density = 1.125gm/lcm3 contalining 0.15M Nact, 10-51 ULA

and 0.02% NaN3 The solution was placed in centrifuge tubes and

underlayered with 5 ml. of KBr solution, density = 1.21gm/cm3; then

centrifuged for 36-48 hrs. at 45,000 RPM using a Ti60 rotor. This

step resulted in the formation of two separate yellow bands; one at

the top of the tube, and one just above the 1.21 plug. The top band

was taken as HDL2 and the lower as HDL The bands were removed using

a Pasteur pipet and the region between the bands discarded. The HDL2

and HDL3 solutions were then dialyzed against 2 x 4 liters of 0.15M

NaC1 containing 10-5% EDTA and 0.02% NaN3, and stored at 4"C until

use. The purity of HDL preparations was checked by SDS gel electro-

phoresis (see next section for reference and details). In no prepara-

tion did the contamination by other proteins (usually serum albumin)

exceed 1-2% and usually no contamination could be seen at all unless

the gels were greatly overloaded.

SDS polyacrylamide gel electrophoresis (SDS-PAGE)

Polyacrylamide gel electrophoresis was carried out using a Model

300B or 301 BioRad Electrophoresis Cell powered by a BioRad Model 400

Power Supply. TLwo types of gel formulation systems were used. First,

gels were prepared using the procedure of Lammli (84). The acrylamide

concentration of the separating gel was adjusted to 10-12% by alteration

of the ratio of acrylamide to water during gel formulation. SDS concen-

tration was maintained at 0.1% and for 5 x 100rmm gels electrophoresis

was carried out at 1.5ma/gel. Bromophenol Blue was used as a tracking

dye and electrophoresis continued until the dye band was approximately

Icm from the bottom of the gel. Gels were fixed in 12.5% TCA: 40%

ethanol: 7% acetic acid, stained with 0.05% Coomassie Blue in 10%

ethanol: 7%. acetic acid, and destained using a Bio~ad Model 172A2

diffusion destainer. Gels were stored in 7% acetic acid.

For reasons to be discussed later, the gel system was changed

during the course of the work to that of Fairbanks, et al. (85). A

7.8% acrylamide gel with 1.0% SDS was prepared and run at 8mal/gel for

5 x 10Dam gels. Pyronin Y was used as a tracking dye. After electro-

phoresis the gels were fixed in 40% propanol: 7.5% acetic, and stained

with 0.05% Coomassie Blue in 40% propanol: 7.5% acetic acid. After

destaining as above the gels were stored in 7.5% acetic acid.

Molecular weights were determined from the recorded scans using

added molecular weight markers as well as the location of the apo-

proteins of known molecular weights.

Crosslinking reactions

The desired amount of protein solution to be crosslinked was added

to a 13 x 100mm glass tube and 10X concentrated buffer added until a

final concentration of 0.025M buffer was achieved. Crosslinker

dissolved in ethanol, water, or acetone (1-5mg/ml) was then added with

rapid mixing to attain the desired molar ratio of crosslinker to protein.

The amount of organic solvent added never exceeded 5% of the total

volume of the reaction mixture. For specific crosslinkers the follow-

ing buffer and solvent systems were used: DFDNB 0.025M Na2CO3 buffer,

pH 8.8, ethanol: DFDNPS 0.025M Na2CO3 buffer pH 8.8, acetone; DMA,

DMSDTB .02M N2HPO4 buffer pH 7.5, water. The reaction mixtures

were incubated for at least 2 hrs. at room temperature unless otherwise

indicated. The reaction mixtures were used directly without further

treatment as it could been shown that only very small amounts of the

free hydrolysis products of the crosslinkers were formed.

For labeling with FDNB, the monofunctional analog of DFDNB, the

conditions described for DFDNB were used.

Amino acid analysis of specific crosslinked products

To determine the amino acid content of a specific crosslinked

product it was first necessary to separate that product from all the

other products of the crosslinking reaction. This was achieved by

preparative gel electrophoresis of fluorescent labeled proteins. Using

a BioRad Model 301 gel system and 10% Lammli gels as described above,

0.5 to 1.0 ml of HDL solution containing 1-3mg/m1 protein, that had

been made fluorescent by reaction with fluorescamine (86), was applied

to the top of each gel. Electrophoresis was carried out at 6 ma/gel

until sufficient resolution of the separate bands was achieved. The

bands were easily visualized using a long wave UV light and after

electrophoresis were sliced from the gel using a razor blade. Slices

from duplicate gels were combined, crushed with a glass rod, and

mixed with 5 to 10 ml of 0.1% SDS solution. The mixture was incubated

at 370C with shaking for 12 hrs., filtered, and the procedure repeated.

The combined solutions were filtered through a .45p Millipore filter

and dialyzed against 3 x 4 liters of H20. The dialysis bag was

agitated to suspend the fine precipitate and the suspension lyophyllized.

The powdered residue was dissolved in 5 ml of 6N-HC1 with 10pl of

B-mercaptoethano1 and 10pl of 0.5% phenol. The solution was sealed in

glass ampules in vacuo and then hydrolyzed for 24 hrs. at 110*C. The

hydrolyzate was dried by lyophyllization, redissolved in 0.2M citrate

buffer pH 2.2, and standard amino acid analysis run on a Beckman

Model 120C amino acid analyzer. Standard citrate buffers and a

ninhydrin detection system were used.

Calculation of the amino acid composition of then proteins analyzed

was carried out using the formula below.

residues of leu or val predicted/molecule XmoeAA=residues AA
mrmoles leu or val molecule

As the amina acid composition was used as a method of verification

of predicted composition, this type of calculation proved most useful.

Delipidation of lipoproteins

Ten ml of HDL solution to be delipidated wereadded with stirring

to 250 ml of chloroform:methano1 (2:1) at -10"C. The turbid solution

was allowed to stand at -10DC for 30 min, 250 ml of cold ethyl ether

were added and the solution filtered on a Buchner funnel through

Whatman #1 filter paper. The filtrate was washed with 250 ml of cold

ethyl ether and dried with a stream of N2. The precipitate was removed

from the paper and stored at -10*C under N2 until use. This method

is a modification of the procedure of Lux et al. (87).

Cel filtration chromatography

Sephadex 0-200 was allowed to swell in water and then poured into

a 2.5 x 100cm glass column to a height of 90cm. It was eqluilibrated

with 0.1% SDS, 0.02M tris-HC1, 10mM NaN3, 1mM EDTA buffer pH( 8.0,

30mg of delipidated protein dissolved in this buffer was added to the

column. An ascending flow rate of 10ml/hr was used and 150 drop frac-

tions collected. The OD28 was monitored using an ISCO UA-2 UV monitor.

The column was stopped after 120ml of buffer had passed through the


Iodination of lipoproteins

Five ml of the sample to be labeled were dialyzed against 0.5M

glycine buffer pH 9.4, containing 0.15M NaC1, 10 M EDTA, and 0.02%

NaN3; then labeled using the iodine monochloride (IM) method of
MacFarlane (88). The iodine carrier solution (0.5ml) containing

250plCi of [12I]-Nal was added to the HDL solution with rapid mixing.

After 10 min. the unreacted iodine and glycine buffer were removed by

gel filtration chromotography using Sephadex G-25 equilibrated withl

0.15M NaC1 containing 10 M EDTA and 0.02% NaN3. Protein solutions

with a specific activity of 5,000 to 10,000cpm/pg were routinely

obtained using this method. Unlabeled controls were treated as above

except for the addition of the [12I]-iodine carrier solution. It

could be shown that approximately 99% of the radioactivity was asso-

ciated with the apoproteins while only 1% was found in the lipid


Incorporation of labeled lipid into HDL,

Fifty mg of celite (50p1 particles, Johns-Mansville Co.) were mixed

with 1.0ml of chloroform to which 50-100pl of labeled lipid dissolved

in toluene-ethanol (1:1) had been added. After mixing, the solvent vol-

ume was reduced with a stream of N2 and the remainder of the solvent

removed in vacuo. Two ml of the HDL solution to be labeled was added

with gentle mixing and the suspension incubated for the desired time

at 370C. The celite was kept in suspension by occasional swirling of

the tubes. After incubation was complete the suspension was filtered

through a 0.45v Millipore filter to remove the celite. All incubations

were carried out for 2 hrs. unless otherwise indicated.

Exch~ange reactions Incubation of HDL2 and HDL mixtures

Thre desired amoulnts of labeled and unlabeled IlDL2 and HlDL3 in

solution were added directly to 5/8" x 3" cellulose nitrate centrifuge

tubes and incubated for the- time and temperature indicated. SDS-PAGE

of controls was used as a method to detect any possible deterioration

of the RDL during incubation. Unless otherwise indicated, for all

incubations described herein the following volumes and concentrations

were used: controls 200pl containing 300pg of HDL protein, mixtures

400pl containing 300vg labeled HDL protein and 300pg cold EDL protein.

For the chaotropic ion experiments, solid KBr was added directly to

the solution to attain the desired concentration of Br ion.

Ultracentrifugation of HDL, HDL, mixtures

The reseparation of labeled and unlabeled HDL2 HDL3 mixtures was

achieved in the following manner. As the mixture to be resolved had

already been placed directly into centrifuge tubes, sufficient NaBr

solution of density =- 1.3288gm/cm3 was added to the mixture to raise

the density to 1.125gm/cm3. NaBr-NaC1 solution (density = 1.125gm/cm3)

containing 0.15M NnC1, 10-5M EDTA, and 0.02% NaN3 was added to give ai

final volume of 10.Dm1. The solutions were then underlayered with

1.0ml of NaBr-NaCL, density 1.21gm/cm3. The tubes were capped and

placed in a Beckman Ti50 rotor for centrifugation at 42,000 RPM for

44-48 hrs.

Gradient Fractionation

After ultracentrifugation all gradients were fractionated using -an

ISCO Model 183 gradient fractionator modified to collect fractions from

the bottom of the tube. Before fractionation each gradient was over-

layered with 1.Dml of 0.15M Nacl solution. 30 drop (0.6ml) fractions

were collected for all experiments.

Activation of Sep~harose by Cyanagen Bromide

Ten ml of packed and well washed Sepharose 4B were placed in a

150ml beaker, 20ml of H20 were added, the pH adjusted to 11.0 with

LM NaOH, and the temperature adjusted to 20'C by the addition of a

small amount of ice. Finely divided CNBr (200mg) were added with

constant stirring and the reaction allowed to proceed until cessation

of proton release was observed by monitoring the pH. The pH was

maintained at 11 during the reaction by the addition of 1M NaOH when

needed and the temperature was adjusted by the addition of ice. When

the reaction was complete, after approximately 15-20 min., 100ml of

ground ice wereadded to the mixture. To Sepharose-ice slurry was then

placed on a coarse glass filter and the Sepharose washed with several

volumes of ice-cold water followed by 200ml of ice-cold 0.1M NaHCO3:

0.5M NaC1 buffer pH 8.0. The washed "activated" Sepharose was used

immediately according to the following procedure.

Coupling of RDL to "Activated" Sepharose

Ten ml of "activated" Sepharose prepared as above or obtained from

Pharmacia were mixed with 10-15ml of 0.1M NaHCO :0.5M Nac1 buffer pH 8.0

in a 100ml beaker. HDL solution (5-10ml of 1-5mg/ml) was added and the

mixture stirred gently for 2-3 hours at 40C. The mixture was then

filtered on a glass filter and washed for 30 min. with several volumes

of IM ethanolamine. The Sepharose-HDL (S-HDL) was then washed with

4 x 100ml of 0.1M NaOAc:0.5M NaCl buffer pH 4.0; 4 x 100ml of 0.1M

NaHCO3:0.5 NaCl buffer pH 8.0; and finally with 2 x 100ml of 0.15M Napl:

10-5M EDTA:0.025% NaN3 The Sepharose prepared in this manner was

stored wet in the final wash solution at 40C.

Exchange reactions using Sepharose-bound HDL

A measured portion of packed Sepharose-HDL labeled with [125] or

radioactive lipid was pipeted into 13 x 100mm glass tubes and an equal

volume of 0.15M NaCZ was added. Cold HDL was added in solution and

the mixture swirled to suspend the gel. After incubation for the

desired time, with occasional swirling to keep the gel suspended, the

mixture was filtered to remove the gel. The filtrate was then counted

in Aquasol-2 in a liquid scintillation counter for lipid exchange exper-

iments, counted in the gamma counter for peptide exchange experiments,

or subjected to ultracentrifugation or SDS-PAGE. For lipid exchange

experiments the following incubation mixtures and times were used:

2591 of Sepharose-HDLg (3mg HDL/ml gel), 200pl of 0.15M NaC1, 50pl of

HDL3 (4tmg/ml) or HDL2' (3mg/ml) incubated for 15 min. at 37"C. For

peptide exchange experiments larger amounts of gel were used due to the

lower levels of HDL binding to the gel (100pg HDL/ml gel). For most

experiments the conditions used were: 400pl Sepharose-[12I]-HDL

(HDL2 or HDL ), 20011l HDL (HDL2 or HDL ) in solution, 200ul 0.15M MaC1.
Incubations were carried out for 5 hours at 370C,

Determination of radioactivity

Al~l counting of [125] was done using a Nuclear Chicago Model 8725

manual gamma counter. The counting efficiency was approximately 63%

based on a comparison with the efficiency of counting of a'Packard

Auto-Gamma Scintillation Spectrophotometer Model 5130 (76% efficiency).

All samples were counted in 13 x 100mm glass tubes using the same volume

of solution in each tube (0.6ml).

For the measurement of [14C] and [ H], liquid scintillation spectro-

scopy was used. The sample was added to 5.0m1 of Aquasol-2 and the


mixture counted in "mini-vials" purchased from New England Nuclear.

All scintillation counting was carried out using a Beckman Liquid

Scintillation Spectrophotometer. The counting efficiency for [14C

was calculated to be 92% while for ( li] the efficiency was 46%.

All scintillation counting values were corrected for background

and counting efficiency by coincidence counting with commercially

available standards.


Isolation and Purity of Lipoprotein Preparations

As the procedure for isolation of HDL is well established (4,6),

the two factors of importance considered were the purity of HDLT prepa-

rations with respect to contamination by other proteins, and the effec-

tiveness of separation during the preparation of RDL subclasses, HDL2

and HDL .

Figures la and lb show the scan of SDS gels of a typical prepara-

tion of RDLT. It can be seen that the only major polypeptide bands

observed have molecular weights that correspond to the major apoproteins

known for HDL. As expected, the ApoA-2 peak (MW 17,500) is split into

its monomer form upon the addition of B-mercaptoethano1 (Fig. lb). The

peak is reduced in size due to a significant loss of low molecular

weight proteins during the fixation and staining of the gels (89).

The purity of HDL2 and HDL3 preparations was checked by recentri-

fugation of the separate subclasses at the density of 1.125gm/cm3 used

during isolation. Under these conditions HDL3 should sink toward the

bottom of the centrifuge tube while HDL2 should float at the top of the

solution. Figure 2 shows the fractionation patterns after this recen-

trifugation. It can clearly be seen that both HDL2 and HDL3 preparations

are pure by the physical criterion of ultracentrifugal mobility. All

preparations of RDL2 were shown to be pure by this method. .HDL~ prepa-

rations, however, occasionally showed some slight contamination by HDL2'

Figure 1. SDS-PAGE of SDS solubilized apoproteins of HDLT

A. Pattern obtained in the absence of reducing agent.

B. Pattern observed when 5% B-mercaptoethanol is
included in the preparation.





Figure 2. Distribution of protein after recentrifugation
of purified HDL2 (6) or HDL3 ($) at a density
of 1.125gm/cm3

1000 +


8 i HDL3 HDL2 *

600 i

400 a

S200 + a

ee e
e? e

0 2 4 6 8 10 12 14 16 18 20


This contamination never represented more than 5% of the total protein

present, and, when observed, an additional centrifugation step was

used to remove it. This HDL2 contamination did not appear to be the

result of HDL3 dimer formation; as once removed, no new contamninant

appeared under any conditions of treatment.

Crosslinking of Total HDL (R~DLT) with DFDNB

The selection of DFDNB as the initial crosslinking reagent to be

evaluated was based on several factors. First, it has been widely

used in previous crosslinking studies involving a variety of protein

types (76). Second, its monofunctional analog FDNB has also been widely

used (76). Third, it was believed that its hydrophobic nature would

'aid in its ability to penetrate the lipoprotein particles. Finally,

pure DFDNB and FDNB are available from commercial chemical laboratories

at low cost.

Figure 3 shows the ge1 patterns observed when a 2mg/ml protein

solution of HDLT is crosslinked withr a 5 fold molar excess of DFDNB

and then run on SDS-PAGE. Figure 3a shows the pattern obtained in the

absence of any reducing agent while 3b is observed in the presence of

1% 8-mercaptoethanol. Although a variety of high molecular weight

crosslinked products are produced (Figure 3a), the first product to

appear and in the highest quantity is a polypeptide complex with an

apparent molecular weight of approximately 46,000. In the presence of

B-mercaptoethanol this major peak is shifted to an apparent molecular

weight of 37,000. Considering only ApoA-1 (HW 28,330) and ApoA-2

dimerr MW 17,500) the appearance of a crosslinked product with a

molecular weight of 46,000 could only be the result of one A-1 linked

Figure 3. SDS-PAGE (10% Lammli gels) of SDS solubilized apoproteins
of HDLT crosslinked with a 5-fold molar excess of DFDNB.

(A) DFDNB crosslinked HDLT, no reducing agent.

(B) DFDNB crosslinked HDLT, with 5% B-mercaptoethanol.







to one A-2 unit. This conclusion is supported by Figure 3b which shows

that the addition of the disulfide cleaving agent, 8-mercaptoethanal,

results in the loss of half of the A-2 dimer leaving a product con-

sisting of one A-1 linked to onle A\-2 monomefr. Th~e molecular weight

of such a complex would be approximately 36,000.

As thle estimation of: molecular weights by SDS get clectrophonresis

can result in considerable error when considering proteins which may

have both inter- and intra-peptide crosslinks (90), additional evidence

is desirable before one can conclude that an A-1***A-2 crosslinked

product is actually produced. To approach this problem it is necessary

to separate the individual crosslinked products so they can be charac-

terized. Gel filtration chromotography using Sephadex G-200 containing

1.0% SDS was attempted. Although this procedure was capable of a

reasonable separation of ApoA-1 from ApoA-2, the crosslinked products

were eluted as a broad, even band; each fraction containing a mixture

of similar molecular weight products. This procedure was not capable

of sufficient purification of any single product. The problem was

solved using the experimental approach diagrammed in Figure 4. The

procedure is detailed in the Methods section of this dissertation. The

results of the amino acid analysis of the 46,000 dalton crosslinked

product is shown in Table 2. Comparison to the predicted values for

sleected amlino acids for an A-1***A-2 complex indicates the 46,000

dalton product is likely a complex of one A-1 and one A-2. Certain

amino acid values were not shown because they were components of the

ge1 buffer system, gave unexpected high or low values for purified A-1

or A-2, or were destroyed during hydrolysis. That both A-1 and A-2

HDL (HDL2 or HDL )

Chemical Crosslinking ----- DS PAGE

Fluorescent Labeling


Slice out protein band

Elution of protein


HCL hydrolysis

Amino acid hydrolysis

Figure 4. Experimental approach to amino acid analysis of
crosslinked products

Table 2.

Amino Acid Analysis of the 46,000 Dalton Crosslinked Product
from HDL, Crosslinked with DFDNB


A-1:A-2 Predicteda










Amino Acid





Aspartic acid











aAll values expressed as residues per mole
for leucine.

of protein based on the value

are components of this complex is suggested by two observations. First,

the shift in molecular weight upon addition of reducing agent could

only occur if ApoA-2 was present as no disulfide bonds occur in Apo-1.

Second, there is no histidine in ApoA-2 or isoleucine in ApoA-1, but

both occur in the 46,000 dalton product. As ApoA-1 contains 5 histidine

residues/molecules and ApoA-2 dimer contains 2 isoleucine residues/mol-

ecule, those must be the source of the histidine and isoleucine found

in the complex. Although the C-peptides contain both isoleucine and

histidine, the low total amount of C-peptides in RDL excludes these

polypeptides from a significant contribution to the composition of any

major crosslinked product.

Effect of Crosslinking on HDL Str'ucture

In discussing the meaning of the results of crosslinking experi-

ments it is important to establish that the introduction of these

crosslinks does not significantly alter the structure of the macro-

molecular complex that is being studied. In the case of HDL, it could

be shown that crosslinked HDL retains the physical properties of native

RDL and will migrate in the analytical ultracentrifuge as a single peak

essentially identical to native RDL. Although even very high levels

of crosslinking (100-fold molar excess) do not appear to disrupt the

particles, all conclusions presented are based on data obtained at

mruch lower levels of crosslinking.

Crosslinking of HDL~ and RDL_ with DFDNB

Although the peptide composition of the two HDL subclasses, RDL2

and RDL appears to be the same, the amount of total protein is

different (1). HDL2 should contain approximately 160,000 daltons of

protein/molecule while HDL3 should only contain 80,000 daltons/molecule.

Under conditions of increasing amounts of crosslinker, one might expect

to observe larger molecular weight crosslinked products for HDL2 as

compared to HDL However,. using the Lammli gel system with molar

ratios of crosslinker higher than 5 to 10-fold, one observes only a

smear of stain at the top of the gel for each subclass. This phenomenon

has been noted for other types of crosslinkers (91). For this reason

a different gel system was used. Fairbanks, et al. have noted that an

SDS concentration of 1%, rather than the 0.1% found in the Lammli

system, is necessary to dissociate membrane protein complexes (85).

Figure 5 shows the comparison of the pattern obtained for HDLT cross-

linked with a 20-fold molar excess of DFDNB and then run on the two gel

systems (5a-Lammli, 5b-Fairbanks). The 1% SDS in the Fairbanks gel

system is apparently sufficient to cause total dissociation of even

highly crosslinked lipoprotein particles.

Figures 6 (HDL2) and 7 (RDL ) demonstrate that for both HDL2 and

HDL3 the crosslinking patterns are essentially the same. It is inter-

esting to note that no crosslinked products of apparent molecular weight

products occur even if the level of crosslinker is increased up to

100-fold. For this reason no separation of HDL2 and HDL3 was carried

out for other crosslinking experiments; instead, HDLT was used.

Crosslinking of HDL_ with DMS and DFDNDPS

To investigate the hypothesis that the hydrophobic nature and length

of the bifunctional reagent used should have some effect .0n the extent

and type of crosslinking produced, a comparison of the gel patterns

obtained was made after DMS or DFDNDPS was used to crosslink HDLT. These

Figure 5. Comparison of the SDS-PAGE methods
of Fairbanks (A) and Lammli (B) for
the separation of highly crosslinked
HDLT (25x DFDNB) .




Figure 6. SDS-PAGE (7.8% Fairbanks gels) of SDS solubilized HDL2
crosslinked with a 10-fold molar excess of DFDNB.


A-2 C


Figure 7, SDS-PAGE (7.8% Fairbanks gels) of SDS solubilized HDL3
crosslinked with a 10-fold molar excess of DFDNB.

A-2 C


two reagents have similar distances between the functional groups but

differ greatly in hydrophobic character. DMS is water soluble and car-

ries a positively charged imido group while DFDNDPS is quite insoluble

in water and must be added in an acetone solution. At low levels of

crosslinker (10-fold molar excess) these two reagents appear to

generate significantly different initial crosslinked products (Figures

8-11). Two major differences between DMS and DFDNDPS crosslinking can

be seen. First, the total amount of crosslinking is smaller for the

hydrophyllic reagent DMS. Second, the initial site of reaction for DMS

appears to differ from DFDNDPS with a 65,000 dalton product appearing

first. At higher molar ratios of these reagents the patterns become

very similar with all different products appearing in similar amounts

in each case.

The initial products produced with DFDNDPS crosslinking are

essentially the same as those formed with DFDNB even though the former

reagent is twice as long as the latter.

Temperature Dependence of Crosslinking

When the crosslinking of HDLT with a 10-fold molar excess of

DFDNB is carried out at different temperatures ranging from 40 to 700C,

no significant differences in the crosslinking patterns are produced.

The number and relative amounts of the separate crosslinked products

remains similar at all temperatures studied. The only difference

observed is a reduced amount of crosslinking at the very high temper-

atures, 60-70"C. It should be noted, however, that the rate of

formation of hydrolysis products of this bifunctional reagent increases

significantly at these elevated temperatures. This would lead to

Figure 8. SDS-PAGE (10% Lammli gels) of SDS solubilized HDL,
crosslinked with a 5-fold molar excess of DMS. No
reducing agent present.





Figure 9. SDS-PAGE (10% Lammli gels) of SDS solubilized HDLT
crosslinked with a 5-fold molar excess of DMS.
5% B-mercaptoethanal included.



Figure 10. SDS-PAGE (10% Lammli gels) of SDS solubilized HDL,
crosslinked with a 5-fold molar excess of DFDNDPS.
No reducing agent present.



Figure 11. SDS-PAGE (10% Lammli gels) of SDS solubilized HDL,
crosslinked with a 5-fold molar excess of DFDNDPS.
5% B-mercaptoethanol included.



decreased availability of reagent as well as an increased probability

of monofunctional labeling.

Interpretation of Cross'linking Results

From the data in this section we conclude that at least some of

the two major apoprotein components must lie very close to one another

in the intact lipoproteln particle since DFDNB will only crosslink

groups about 5A apart. If each HDL particle contains both A-1 and A-2

molecules, a number of possible crosslinking products could result.

From our findings with low levels of DFDNB (5-10 fold molar excess)

several observations can be made. First, the major crosslinked product

appears to comprise one A-1 plus one A-2; second, there is no indication

of any A-2 intramolecular crosslinking which might be expected with a

molecule already crosslinked with a disulfide bridge. .There also

seems to be little significant crosslinking-reactive association of two

A-1 molecules as suggested by Scanu (92). Since both A-1 and A-2

contain numerous reactive residues fairly evenly distributed throughout

their sequence (1), any close association of the chains should make

multiple sites available for possible crosslinking.

Experiments using higher molar ratios of crosslinker (20-100X) do

show that most possible combinations of A-1 and A-2 are formed. Still,

even at these high concentrations, no A-2 intramalecular crosslinked

molecules are produced. The C-peptides do not appear to be involved in

the crosslinking reactions. At high levels of reagent the amount of

unreacted C-peptide does not appear to decrease when compared to con-

trols. This suggests little association of C-peptides with A-1 or A-2.

The formation of most of the possible products at high crosslinker

concentrations seems to indicate either a significant degree of mobility

of the apoproteins or significant alteration of the native structure of

the particle. 2Two factors suggest that although some mobility exists,

a high degree of specific association of the apoproteins occurs in the

intact HDL particle. First, the decreased crosslinking observed at

high temperature could be due to increased mobility of the polypeptide

chains. Second, the failure of A-2 to form intramalecular crosslinks

suggests a stable conformation that remains fairly fixed even at these

elevated temperatures. The reproducibility of the crosslinking exper-

iments with respect to order of formation and ratios of products formed,

even when HDL is obtained from varied sources, suggests that in the

intact HDL particle the apoprotein components may be found in a rather

fixed spatial orientation to one another.

That the crosslinking patterns for HDL2 and RDL3 do not differ also

argues for a fixed association of 2A-1 and 1A-2 apoproteins. Even though

RDL2 may have two HDL3 protein subunits (54), these do not appear to

interact. This would lead one to speculate that HDL2 might be a dimer

of 2-RDL3 molecules. This idea will be discussed in detail later in

this work.

An additional argument in favor of fixed associations with flexi-

bility of the apoproteins comes from the experiments using the two

reagents DMS and DFDNDPS. These two reagents consistently yield initial

crosslinking products that.differ. This may occur due to different

initial sites of reaction for these reagents. The initial products do

not differ when one compares DFDNB and DFDNDPS, although the latter is

twice as long as DFDNB. This again suggests some flexibility in the

associations of the apoproteins.

From these observations one might suggest a model in which the

apoproteins lie in a fixed, flexible conformation held in place in

part by protein-protein, lipid-protein, and lipid-lipid interactions.

The ability to bind lipids may in turn be governed by-these inter-

actions; the flexibility allowing for differing degrees of binding of

specific "functional" lipids while certain "structural" lipids remain


Aorotein Exchange Between HDL^ and RDL

If the ratio of A-1 to A-2 dimer is 2:1, in both HDL2 and HDL3 as

suggested by Friedberg and Reynolds (54), then the difference between

RDL2 and HDL3 may represent the addition of one A-1:A-2 unit, which
consists of two A-1 plus one A-2 peptide dimer. Because our cross-

linking data suggests a degree of flexibility as well as specific inter-

action within an A-1:A-2 unit but the two units do not appear to cross-

link in intact HDL2, it is necessary to consider possible modes of

association of the apoproteips of the two proposed units in HDL .

To investigate this possible association, as well as the relation-

ship between the apoproteins of the different subclasses of HDL, we

have examined the exchange of labeled apoproteins between HDL2 and HDL3,

in the presence and absence of bifunctional crosslinking reagents.

Figures 12 and 13 show the ultracentrifugal fractionation patterns

obtained for controls (300ug HDL protein in 200pl) of [12I]-labeled

HDL3 and [125I-labeled HDL2 incubated In the absence of any other
protein. The results are expressed as dpm per fraction as it was found

that the radioactivity and protein concentration curves coincided (the

specific activities of control fractions were constant throughout the

Figure 12. Exchange of apoproteins between [12I]-HDL3 and HDL2'

Distribution of label after centrifugation tosaat
HDL2 and HDL3. $ = [125I]-HDL3 control; O =[1 ]HD
incubated with cold HDL2*

20000 +

[ I]-HDL
16000 + 3

+ HDL2

12000O +

8000 +B 8


0 2 4 6 8 10 12 14 16 18 20


Figure 13. Exchange of apoproteins between [1250-HDL2 and HDL3.

Distribution of label ~ter centrifugation to a Parate
HDL2 and HDL3. $ = [1 I]-HDL2 control; 8 = [1 DI]-HDL2
incubated with cold HDL3

40000 + e

32000 +

24000 +-

~ [125]-HDL2
16000 +F21
~ + HDL3

8000 + /"a.

0 2 4 6 8 10 12 14 16 18 20

tube). Figures 12 and 13 also illustrate the results obtained after

mixing equal amounts (300pg protein in 200)11) of (12I]-labeled HDL3

and cold HDL2 (Figure 12) or (12Ii-labeled HDL2 and cold HDL3 and

incubating the mixture for 7 brs. at 370C (Figure 13). In Figure 12

it can clearly be seen that A transfer of label from HDL3 to HDL2 has

occurred. Further, the specific activity of the HDL3 fraction was

significantly decreased when compared to controls. In a typical

experiment, the values for a selected fraction in the RDL3 region of

the tube (fraction 6) were: [12I]-HDL3 control = 2850 dpm/pg;

112I]-HDL3 incubated with cold HDL2 = 1680 dpm/pg. Similar results

were obtained when labeled HDL2 was incubated with cold HDL3 (Figure 13).

However, due to the breadth of the HDL3 peak as compared with the HDL2

fractions, the results are not as easily visualized.

Effect of Crosslinking and Monofunctional
Labeling on Apoprotein Exchange

If the labeled HDL3 is crosslinked with DFDNB before incubation

with HDL2, the transfer of labeled protein is reduced depending upon
the extent of crosslinking. Figure 14 shows the three fractionation

patterns obtained with : [12I]-HlDL3 incubated with HDL2 (control);

[125I]-HDL3j crosslinked with a 5-fold molar excess of DFDNB and then

incubated with HDL2; and [12I]-HDL3 incubated with HDL2 following

crossl~inking of the HIDL3 with a 25-fold molar excess of DFDNB. It canl

be seen that increased crosslinking results in decreased exchange.

To rule out the possibility that the monofunctional substitution

of the apoproteins with the crosslinking reagent was the cause of this'

decrease in exchange, FDNB, a monofunctional analog of DFDNB, was used

to modify the proteins. Reaction with concentrations of FDNB, comparable

Figure 14. Effect of crosslinking on the apoprotein exchange between
[125IJ-HDL3 and HDL2

Distribution of label after centrifugation to separate
HDL2 and HDL3. 6 = [125I]-HDL3 incubated with cold HDL2;
$ = [125I] _"L3 crosslinked with a 5-fold molar excess of
DFDNB followed by incubation with HDL2; P = [125I]-HDL3
crosslinked with a 25-fold molar excess of DFDNB then
incubated with HDL2*

20000 +

16000 +

12000; +

soo e e se~e e

4000 + \

0 +********..... ***********************--*+ **.......+....... .. ......4......... ....+

O 2 4 6 8 10 12 14 16




18 20

to amounts of DFDNB that greatly inhibited exchange, had little effect

on the exchange process (Figure 15).

When HDL2 is crosslinked before incubation with labeled llDL the

exchange process is also Inhi~bited (Figulre 16). 'The reverse also hioldu

true; that is for crosslinked HDL3 incubated with labeled HDL2, the

exchange is reduced.

Effect of Temperature and Dilution on Peptide Exchange

Both low temperature and dilution have an effect upon the rate of

protein exchange in HDL. If the incubation is carried out at 4.0, very

little exchange will occur in the time period in which significant

exchange occurs at 37"C (Figure 17). Using the amounts of HDL2 and

HDL3 described in the Methods section, but diluting this mixture with
a 10-fold excess (5ml) of 0.15M NaC1, the rate of exchange at 370C is

also significantly reduced (Figure 18). These two observations are

consistent with a mechanism of exchange involving a collisional com-


It is this reduction in exchange rates that permits one to employ

the 48 hour centrifugation requir-ed for reseparation of the HDL2 and

HDL3 mixtures. When equal amounts of labeled HDL3 and cold HDL2 are

mixed, then immediately diluted, cooled to 4.0, and placed in the

ultracentrifuge, essentially no exchange occurs, even after 48 hrs.

Rate of Peptide Exchange

Figure 19 shows a time course plot for the exchange of apoproteins

from [12I]-HDL3 into HDL2. The radioactivity represents the total

transferred into the HDL2 region of the centrifuge tube after subtrac-

tion of the amount in those fractions attributed to HDL Estimation

Figure 15. Effect of monofunctional labeling with FDNB on the exchange
of apoproteins between [125I]-HDL3 and HDL2*

Distribution of label after centrifugation to separate HDL2
and HDL~3. 6 = [125I]-HDL3 reacted with a 25-fold molar
excess of FDNB; 8 = [125I]-HDL3 reacted with a 25-fold molar
excess of FDNB followed by incubation with HDL2*

00 +

00 +






[12I]-HDL3 25X FDNB

[125 L3 25X FDNB

/ p + HDL2

0/ +

,0 + t
tee *

o +*********+. - -----.:........+......~...+..... ...........+.. .........4.......,.. ......... ... _~

0 2 4 6 8 10 12 14 16 18 20

Figure 16. Effect of crosslinking the unlabeled HDL subclass on
the exchange of apoproteins between [1251]-HDL3 and HDL2.

Distribution of label after centrifugation to separate
HDL2 and HDL3' O = 125I]-HDL3 control; 8 = [12b] -HDL3
incubated with HDL2 crosslinked with a 25-fold molar
excess of DFDNB.

20000 +

16000 +

12000 +F4

4000 125I]3-HDL3

rl5]-HDL + HDL2 25X DFDNB

0 +*********+********+********4 *******.+........-t +.................4 c...... ...4.e ....

0 2 4 6 8 10 12 14 16 18 20

Figure 17. Effect of temperature on the apoprotein exchange between
[125I] HD 3 and HDL2
Distribution of label after centrifugation to se rate
HDL2 and HDL3. 9 = [125I]-HDL3 control; af = 12IJ-HDL3
incubated with HDL2 at 4oC. Compare to Figure 12.

20000 +


12000 + /

sooo +

0~ * + * * * * . . . . . + . . 4 . . . . o . g
a 0 0 2 4 8 1 1 4 6 18 2

Figure 18. Effect of temperature and dilution on the apoprotein
exchange between [125I]-HDL3 and HDL2*

Distribution of label after centrifugation to separate
HDL andHDLg 6= 125I]-HDL, and HDL, were incubated
at C after dilution with 5ml30f 0.15M2NaCl.

20000 +

16000 +

12000 +F

8000 +


e e-

e' e

e a

0 +*********+*********+***-*****+ ***. .....+.........'.. ................4........4........4.......

0 2 4 6 8 10 12 14 16 18 20

Figure 19. Time course for the exchange of [ I]-labeled apoproteins
from [125I]-HDL3 into HDL2. 3mg HDL protein/ml each subclass.

6500 +

5200 +
e a

/ 3900; +


a1300 +

O 36 72 108 144 180 216 252 288 324 360


of the initial slope at 2300 dpm/hr out of a total of 90,000 dpm gives

an exchange rate of approximately 2.5%/hr. These calculations are based

upon two separate experiments utilizing concentrations of llDL.2 and

HDL3 companrable to plasma levels (3mng/ml eachc subclass). Th'le rate

can be altered by varying the initial concentrations of either or both


Effect of Bromide lon on Protein Exchange

If the chaotropic Br ion is included in the.incubation mixture at

37PC, the amount of exchange in a given time period is greater when

compared to controls with no Br Figure 20 shows the fractionation

pattern obtained when 2M1 KBr is included in the 7 hour incubation solu-

tion as compared to controls. A significant increase in exchange can

be seen. As chaotropic ions are known to effect protein conformation

and association (93), these factors may be involved in the exchange


SDS-Polyacrylamide Gel Analysis of Exchanged Proteins

Gel electrophoresis of the recipient HDL subclass, into which

labeled apoproteins have been transferred, indicates that ApoA-1,

ApoA-2, and the C-peptides are all exchanged. Figure 21 shows the

radioactivity pattern obtained when RDL2, into which labeled apopro-

teins have been transferred by incubation with [ 2I]-HDL3, is run on

SDS-PAGE. The radioactive bands were identified by staining and

scanning duplicate gels.

If the labeled HDL subclass is subjected to limited crosslinking

with a 10-fold molar excess of DFDNB before mixing and incubation

Figure 20. Effect of KBr on the exchange of apoproteins between
[125I]-HDL3 and HDL2

Distribution of label after enetrifugation to separate
HDL2 and HDL3. 6 = [125I]-HDL3 incubated with HDL2;
s = [1251]-HDL3 incubated with HDL2 in the presence of
2M KBr.

50000 +

40000 +



10000 + e *

0 ****+**********+ **** + **********+ ****+ ******** *****

100 2,B / 0 2 1 1 8 2


Figure 21. Polyacrylamide gel analysis of exchanged apoproteins.
SDS-PAGE of HDL2 after incubation with [ I]-HDLa
followed by reseparation. The gel was sliced it
2mm slices and the slices counted.

2500 +

2000 +


W,1500 +

e ~6

0 4 8 12 16 20 24 28 32 36 40


with cold HDL, the labeled apoproteins that are exchanged are those

that were not crosslinked. Figure 22 shows the gel pattern obtained

for crosslinked [12I]-HDL3 alone, and for HDL2 that was labeled by

exchange of apoproteins from crosslinked [125]-HDL Although a

significant portion of the HDL3 apoproteins were crosslinked into

higher molecular weight products, only uncrosslinked A-1 and A-2

appear to be exchanged.

Interpretations of Protein Exchange Experiments

The transfer of labeled apoprotein from one HDL subclass to

another does not proceed with a loss of protein content of the orig-

inally labeled fraction. The specific activity of that fraction

decreases but the protein content remains unchanged. This is con-

sistent with an exchange process in which there is a one to one exchange

of protein of one subclass with the other. Such exchange could occur

by the transfer of single polypeptide apoproteins, i.e., A-1 or A-2;

or by the exchange of complexes of apoproteins such as 2A-1:1A-2.

These experimental results do not permit us to rule out either of these

possibilities. It might be suggested that since crosslinking inhibits

the exchange, single polypeptide chains are involved. However, the

crosslinking may also prevent a conformational change that might be

necessary for the exchange process. That a conformational change

might be involved is suggested by the fact that when high concentra-

tions of the chaotropic ion, Br are included in the incubation mixture,

increases in the rates of exchange are observed. An actual exchange

involving a conformation change is also suggested by the finding that

crosslinking the unlabeled subclass also greatly inhibits the exchange

Figure 22. Polyacrylamide gel analysis of exchanged apoproteins:
Effect of crosslinking with DFDNB.

SDS-PAGE of HDL2 reisolated after incubation with [ IJ-HDL3
crosslinked with DFDNB. Q = [125I]-HDLgL crosslinked with a3
25-fold molar excess of DFDNB; 6 = HDL2 after incubation with
crosslinked [125I]-HDL3.

400 +

320 +

240 + I

S160 +

*:~ ~ h,

0~~t +* *******.....+ .... ....... s

O 4 8 12 16 20 24 28 32 36 40

process. If a simple transfer of peptide into the recipient class

occurred, one would not expect crosslinking to have much effect.

If HDL2 is made up of two identical subunits as suggested by

Friedberg and Reynolds (54), the transfer of label might be visualized

in terms of the exchange of RDL3 for one of the HDL2 subunits.

Although this is a reasonable possibility, one must again consider the

possibility of a conformational change to explain the results of the

crosslinking experiments.

Even though the two RDL subclasses can exist as stable separate

entities, when they are present together in solution, significant inter-

action between particles may occur. Exchange of apoproteins could

possibly occur between HDL2-HDL2 or HDL3-HDL3, as well. as between HDL2

and RDL3 molecules. If collisional complexes cause exchange and single

polypeptides are capable of exchange, then one would expect HDL -HDL3

exchange to occur. Collisional complex formation should also result

in the rapid exchange of lipids of all types due to their small size

and the fusion required for peptide exchange.

Lipid Exchange Between HDL2 and HDL, in Solution

Whcn IlDL3 Is Ioledcir withl [310j-chleslctcorl, [3Hl]-cholesterol panlm--

tate, or (14C]-phosphatidyl choline, thecn incubated with cold HDL2, as

withi peptide exchange; label is transferred to HDL2. However, even

under conditions of low temperature, dilution, and no incubation, total

exchange occurs during the ultracentrifugal reseparation of R1DL2 and

HDL3. This rapid movement prevents the study of the exchange using the
experimental approach that was possible for peptide exchange. For this

reason an alternative approach was developed.

Binding of Labeled HDL to Sepharose

The rapid rate of lipid exchange places one requirement on the

method employed to study this exchange: the separation of HDL3 and

HDL2 'must be done very quickly. As use of the ultracentrifuge requires
at least 24 hours even at 60,000 rpm, it was decided to approach the

problem by immobilizing one of the subclasses by covalently binding

it to Sepharose. This would allow separation by filtration; a process

that would take less than one minute.

When (12I]-HDL3 was reacted with commercially obtained CNBr-acti-

vated Sepharose 4B (Pharmacia) under the coupling conditions described

in the Methods sections, a Sepharose preparation containing approxi-

mately 0.5mg protein per ml of packed Sepharose was obtained. To

determine what percentage of the protein was bound to the Sepharose, a

portion of the gel was washed with 1% SDS solution. Under these

conditions any protein chains not covalently bound to the gel should

be washed off. Table 3 shows the results of such an experiment. Using

the Pharmacia CNBr-activated Sepharose, no more than 30% of the label

could be removed with SDS. Assuming only 3 polypeptide chains/HDL3

molecule, this indicates that an average of two are linked to the

Sepharose. To obtain a preparation with a lower level of binding, it

was necessary to activate our own Sepharose 4B using the conditions

described in the Methods section. Under these conditions, preparations

with over 50% of the protein removable with SDS could be obtained. The

same conditions also gave acceptable levels of binding for HDL2 prepa-

rations (20-40% bound). For all peptide exchange experiments,

preparations with low levels of binding were used. For lipid exchange

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