NBSIR 74-470

Interaction of Plasma Proteins with

Surfaces

C. A. Fensterniaker, W. H. Grant, B. W. Morrissey, L. E. Smith, R. R. Stromberg

PolyiTieib Oivisioii

Institute I'uf Materials Research

National Bureau of Standards

Washington, D. C. 20234

March 22, 1974

Annual Report tor Period

July 1, 1972 -November 30, 1973

Prepared tor. Biomaterials Hiogram

Division of Blood Diseases and Resources

National Heart and Lung Institute

National Institutes of Health

Bethesda, Maryland 20014

Interagency Reimbursable Agreement

NBSIR 74-470

INTERACTION OF PLASMA PROTEINS WITH

SURFACES

C. A. Fenstermaker, W. H. Grant, B. W. Morrissey, L. E. Smith, R. R. Stromberg

Polymers Division

Institute for Mrterials Researcli

National Bureau of Standards

Washington, D. C. 20234

March 22, 1974

Annual Report for Period

July 1, 1972- November 30, 1973

Prepared for. Biomaterials Program

Division of Blood Diseases and Resources

National Heart and Lung Institute National Institutes of Health

Bethesda, Maryland 20014

Interagency Reimbursable Agreement

U. S. DEPARTMENT OF COMMERCE, Frederick B. Dent. Secretary

NATIONAL BUREAU OF STANDARDS, Richard W. Roberts. Director

BIBLIOGRAPHiC DATA

SHflET NBSTR 74-470 . 4. i 11 ii.- ar..'. >i:ii; it Interaction of Plasma Proteins with Surfaces

3. Ktcipicnt's :\

March 22, 1974

6.

7. Author{=.) Fenstermaker, W. H. Grant, B. W. Morrissey,

L. E. Smith and R. R. Stromberg

5. l-'crf orm ing Organizacion Ki fi NBSIR 74-470

9. Pcrlorniin;; Org a n i ^.at i on Nai;ic and AJdrL-ss National Bureau of Standards

Department of Commerce

Washington, D. C. 20234

10. I'r o|Cc t / 1 as k, 'A ork L'nit .\ c

11. C^ontract 'Cirant No. Interagency Reimbursable Agreement

12. >ponsorin_c Orf'.ani/ation Xaine and Address Biomaterials Program, Thrombosis & Hemorrhagic Diseases Branch

Division of Blood Diseases & Resources, Nat. Heart & Lung Inst,

NIH, Bldg. #31, Room 4A05

Bethesda, Maryland 20014

13. 1 ype ot Ktpori & Period Covered July 1, 1972

Nov. 30, 1973

14. NHL I

15. Supplc-nientiiry Notes

16. Abstracts The interaction of blood proteins with surfaces has been investigated with principal

attention focused on those proteins that are either major constituents of blood plasma

or are implicated as being important in the clotting process. Emphasis has been placed

on molecular conformational changes occurring upon the interaction of such proteins with

surfaces. The extension of adsorbed molecules of fibrinogen, albumin, and prothrombin

on a number of selected materials vjas studied by ellipsometry . The results indicate a dependence of conformation on surface energy. Measurements of the bound fraction

(number of carbonyl surface attachments) of these adsorbed blood proteins on a silica

surface showed that approximately ten percent of the carbonyl groups were attached to

the surface for prothrombin and serum albumin at all values of surface population for

the solution concentrations studied. Competitive interactions of prothrombin and

fibrinogen during the process of adsorption, displacement, and desorption have been

measured and rates of adsorption of albumin were measured on chrome and silica surfaces.

17. Key Uords and Documeni .Analysis. 17a. Descriptors

Adsorption; blood protein; bound fraction; ellipsometry; polymer adsorption; protein

adsorption

17b. Idcntif icrs/Open-Ended Terms

17c. COSATl I-ield/Group

18. Avail abii ity Statement Release Unlimited - Available from:

National Technical Ir.f or'ination Service

I 528 5 Port 3.oy.?.l Road, Springfield, Va. 22151

19. Security Class (Ihis

Report

)

fNCl-ASSIFlKD

21. No. ol Pajtes

72

20. Security Class ( This

i;xr-i,AssiF!!-n

22. 1-nce

Tills rOi'.M MAY BE REPROOUCh <M-DC M 352-f

TABLE OF CONTENTS

Page

ABSTRACT i

SUMMARY ii

INTRODUCTION 1

METHODS 3

Ellipsometry 3

Infrared Difference Spectroscopy 6

Computer Simulation 15

Radiotracer 15

EXPERIMENTAL 18

Ellipsometry 18

Infrared Difference Spectrometry 19

Radiotracer 20

RESULTS AND DISCUSSION

MOLECULAR EXTENSION 22

Time Dependent Behavior 22

Equilibrium Values 32

BOUND FRACTION 42

Equilibrium Conformation Studies 42

Effect of Surface Population 42

Variation of pD and Ionic Strength 48

Non-Equilibrium Conformation Studies 50

Dependence of Conformation on Adsorption Time 50

Comparative Rates of Adsorption 50

Changes in Conformation of Serum Albumin Upon Adsorption. . 53

Conformation Studies of Model Compounds 56

Experimental and Computer Simulation of Lysozyme

Adsorption 56

Polyamino Acid Studies 58

EFFECT OF ADSORPTION ON THE THROMBIN-FIBRINOGEN REACTION. ... 59

COMPETITIVE ADSORPTION WITH UNLABELED PROTEINS 59
TABLE OF CONTENTS (cont.)

Page

RESULTS AND DISCUSSION (cont.)

RADIOTRACER RATE STUDIES 64

Development of Washing Techniques 64

Surface Roughness Determinations 65

Adsorption Rate Results 66

Chromium 66

Silica 66

Platinum 66

REFERENCES 72
FIGURES

Page

Figure 1: Schematic representation of reflection from film

covered surfaces ^

Figure 2: Schematic representation of ellipsometer 7

Figure 3: Photograph of ellipsometer 8

Figure A: Photograph of ellipsometer stage with cell 9

Figure 5: Schematic diagram of adsorption cell 10

Figure 6: Shift in absorption frequency of amide I band as a result of carbony] group interaction with surface ... 11

Figure 7: Experimental method for infrared difference

measurements 13

Figure 8: Infrared difference spectrum of bovine serum albumin

adsorbed on silica 14

Figure 9: Schematic representation of lysozyme adsorbed on planar surface 16

Figure 10: Extension of fibrinogen on chromium 23

Figure 11: Adsorbance of fibrinogen on chromium 24

Figure 12: Extension of prothrombin on chromium 25

Figure 13: Adsorbance of prothrombin on chromium 26

Figure 14: Extension of prothrombin on laminar carbon 27

Figure 15: Adsorbance of prothrombin on laminar carbon 28

Figure 16: Extension of prothrombin on polyethylene 29

Figure 17: Adsorbance of prothrombin on polyethylene 30

Figure 18: Adsorbance of albumin on chromium 31

Figure 19: Adsorbance of Cohn Fraction I on laminar carbon .... 33

Figure 20: Extension of Cohn Fraction I on laminar carbon 34

Figure 21: Adsorbance of Cohn Fraction I on polyethylene 35
FIGURES (cont.)

Page

Figure 22: Extension of Cohn Fraction I on polyethylene 36

Figure 23: Adsorbance of Cohn Fraction I on polyethylene with

fibrinolysin added 37

Figure 24: Adsorbance of Cohn Fraction I on polyethylene with

£-aminocaproic acid added 38

Figure 25: Adsorbance of purified fibrinogen on polyethylene ... 39

Figure 26: Adsorption isotherm for bovine serum albumin at pD

7.4 on silica 43

Figure 27: Adsorption isotherm for prothrombin at pD 7.4 on silica

Figure 28: Adsorption isotherm for bovine fibrinogen at pD 7.4 on silica 45

Figure 29: Bound fraction vs. adsorbance for bovine fibrinogen

at pD 7.4 on silica 46

Figure 30: Adsorbance vs. pD for bovine serum albumin on silica

for several ionic strengths 49

Figure 31: Adsorbance and bound fraction vs. time for bovine serum

albumin, prothrombin, and fibrinogen 51

Figure 32: Adsorbance vs. time for 1.75 x 10 ^ M bovine serum

albumin, prothrombin, and fibrinogen 52

Figure 33: Computer simulation of lysozyme adsorption - Number of

carbonyl oxygen atoms vs_, distance for the adsorbed

conformation , 57

Figure 34: Competitive adsorption of unlabeled serum albumin

and fibrinogen 60

Figure 35: Competitive adsorption of unlabeled serum albumin

and fibrinogen 61

Figure 36: Displacement of serum albumin by fibrinogen 62

Figure 37: Desorption of serum albumin 63

Figure 38: Rate of albumin adsorption on chromium 67
FIGURES (cont.)

Page

Figure 39: Adsorbance of albumin on chromium by ellipsometry . . . 68

Figure 40: Adsorption isotherm of albumin on silica . . 69

Figure 41: Rate of albumin adsorption on silica 70

TABLES

Page

Table I: Ellipsometric Adsorbance and Extension Data on Various Surfaces 41

Table II: Summary of Adsorption and Bound Fraction Data for

Proteins 47

Table III: Fraction of Protein Adsorbed 54

Table IV: Bound Fraction of Modified Serum Albumin 55

Interaction of Plasma Proteins with Surfaces

Abstract

The Interaction of blood proteins with surfaces has been investigated

with principal attention focused on those proteins that are either major

constituents of blood plasma or are implicated as being Important in the

clotting process. Emphasis has been placed on molecular conformational

changes occurring upon the interaction of such proteins with surfaces. The

extension of adsorbed molecules of fibrinogen, albumin, and prothrombin on

a number of selected materials was studied by ellipsometry . The extension

was found to Increase with decreasing surface energy for the surfaces studied,

although the amounts adsorbed from solutions of physiological concentrations

remained approximately constant. These results indicate a dependence of

conformation on surface energy. Measurements of the bound fraction (number

of carbonyl surface attachments) of these adsorbed blood proteins on a silica

surface showed that approximately ten percent of the carbonyl groups were attacnea

to the surface for prothrombin and serum albumin at all values of surface

population for the solution concentrations studied. Fibrinogen showed an increasing bound fraction with increasing adsorbance. The fact that no change in bound fraction occurred upon prior cross-linking and denaturation

of albumin implied that changes in conformation upon adsorption, if they

occurred, were small. Competitive interactions of prothrombin and fibrinogen

during the process of adsorption, displacement, and desorption have been

measured and rates of adsorption of albumin were measured on chrome and

silica surfaces.

1
Interaction of Plasma Proteins with Surfaces

SUMMARY

The interaction of blood proteins with surfaces has been investigated with

principal attention focused on those proteins that are either major constituents

of blood plasma or are implicated as being important in the clotting process.

Emphasis has been placed on molecular conformational changes occurring upon

the interaction of such proteins with surfaces. Materials have been selected

which provide an insight into the mechanisms of these interactions. Materials

for future study will include those which have been used or show potential

utility in surgical procedures.

The extension of adsorbed molecules of fibrinogen, albumin, and prothrombin

on a number of selected materials was studied by ellipsometry . No change in

extension was observed for a given surface over a time period during which

adsorbance was found to increase. This indicates a lack of molecular rearrangement after initial adsorption. The equilibrium extension was found to increase

with decreasing surface energy for the surfaces studied, although the amounts

adsorbed from solutions of physiological concentrations remained approximately

constant. These results indicate a dependence of conformation on surface energy.

Measurements of the bound fraction (fraction of carbonyl groups of an adsorbed molecule directly in contact with the surface) of these adsorbed blood

proteins or, a silica surface showed that appToxim=i*"p1 y ten percent of the carbonyl

groups were attached to the surface for prothrombin and serum albumin at all values

of surface population tor the solution concentrations studied. Fibrinogen

showed an increasing bound fraction with increasing adsorbance. The fact that

no change in bound fraction occurred upon prior cross-linking and denaturation

of albumin implied that changes in conformation upon adsorption to the silica

surface, if they occurred, were small. Computer simulation studies of the

adsorption of lysozyme were found to be consistent with these findings. Studies

of the bound fraction of serum albumin as a function of pD and ionic strength

show that caution must be exercised in identifying the plateau value for an adsorption isotherm for a protein with a close-packed monolayer.

Techniques have been developed for use with infrared spectroscopy which

take advantage of the unique biological properties of prothrombin and fibrinogen

to study competitive interactions of these proteins during the process of

adsorption, displacement, and desorption. Some results are given.

Radiotracer studies have been initiated for measurements of the rates of

adsorption and desorption. Washing techniques for such investigations have

been developed. Equilibrium results obtained by the radiotracer method have

been found to compare favorably with those obtained by ellipsometry. Rates of

adsorption for albumin on chrome and silica surfaces have been measured. An

important artifact was observed in the attempted adsorption measurements using

iodine labeled albumin on a platinum surface. Specific adsorption of iodine causes preferential adsorption of the labeled protein.

I

ii
INTERACTION OF PLASMA PROTEINS WITH SURFACES

INTRODUCTION

At present, the surface-induced coagulation of blood is a critical

factor in the design and application of most devices for use with the

cardiovascular system. The original studies of Freund (1) in 1885, as well as much recent work, e.g. ref. (2,3), have shown that the clotting

time of blood is dependent upon the materials with which it is in contact.

The current cascade model (4) emphasizes the role of numerous proteins in an enzyme-substrate relationship and stresses the probability that the initial

event of surf ace- induced coagulation involves an interaction between the

surface and a protein. The possible effects of a given surface on a protein

would include, among others, permanent or reversible adsorption with or without concomitant denaturation and preferential adsorption of specific

proteins. It is, therefore, of great importance to elucidate the details

of the interaction of blood proteins with surfaces, especially if one hopes to develop a rational approach to the selection of thromboresistant

materials

Investigations on the interaction of blood proteins with surfaces have

been recently reviewed (5-8) . These studies have attempted to correlate the

extent of the coagulation of plasma or blood with such surface properties as surface free energy (2,9), roughness (10), surface charge (11), and zeta

potential (12). With few exceptions (13), investigations of the specific

interaction of an adsorbed protein on a surface have, in general, been carried

out on dried, collapsed films. Hence, at present, very little information

exists that would enable a specification of the state of an adsorbed protein

in situ . The importance of this information is stressed by recent studies (14)

showing no patterns of selective adsorption or depression of enzymatic activity

of blood factors by a number of surfaces when investigated by analysis of

the supernatent solutions.

There is a need for a detailed understanding of the major events

occurring at the blood-implant interface in order to assess their con- sequences and thus provide a rational basis for the design of materials and

test methods. To this end we have studied various aspects of the interaction

of several blood proteins with suitable surfaces, utilizing methods which

have proven successful with synthetic polymer systems. Our attention has

been focused initially on those proteins that are either major constituents

of blood plasma or are implicated as being important in the clotting process.

The surfaces investigated are those concerned with materials which have

been used or show potential utility in surgical procedures or those which

provide an insight into the mechanisms of the interactions. It is anticipated

-1-
that this information will be of immediate value in the selection of

materials for use in heart assist devices such as catheters, heart valves,

blood oxygenators, etc.

The specific aspects of the protein surface interactions that we have

been investigating can be separated into three general areas:

1) Aspects of the changes in the conformation of the protein upon

interaction with a surface. Specifically we are studying the

changes in the average dimensions of the protein molecules as they interact with a surface as well as the number of contacts

the molecule makes with the surface.

2) Measurement of the rates and amount of adsorption and desorption

of individual proteins. We are also interested in the competitive

nature of these processes, e.g., the adsorption of two or more

proteins simultaneously or the replacement of an adsorbed protein

by another.

3) Determination of the effect of adsorption on the reaction of

enzyme-protein systems. This includes studies of the effects

of adsorption on the enzyme, the substrates, and on any end

product.

-2-
METHODS

Three distinct techniques have been employed in this investigation

and are described separately below in some detail.

Ellipsometry

Ellipsometry is quite unique in its ability to give a measure of the

average dimensions of an undisturbed protein layer adsorbed on a surface.

Briefly, ellipsometry is an optical technique in which the change in state

of polarization of light upon reflection from a surface is used to characterize

the surface and to determine the thickness and refractive index of a thin

film overlaying that surface. The thickness and refractive index of this

film can be determined simultaneously from these measurements. From these

independently determined quantities, one can also determine the concentration

of protein in an adsorbed film as well as the amount of protein adsorbed

on a surface. The technique has the advantage that measurements can be

made in situ , that is, during the time required for interactions and changes

in molecular shape and without introducing any artifacts due to removal of

the surface from the solution.

A wide variety of sample types, both metallic and dielectric, may be

studied. The only general requirements are that the surface or interface

be well defined and smooth enough to give specular reflection and that it

be stable under the experimental conditions. The sensitivity of the mea- surement is dependent upon the details of the experimental system, mainly

the refractive indices of the solution, film, and surface involved; films

as thin as a few angstrom units can be measured under the proper conditions.

A typical system for study by the ellipsometer consists of a film of

index n2 and thickness d on a reflecting substrate of index n3 immersed

in a medium of index n^, as shown in Figure 1. Let all media be isotropic

and ni represent a real index of refraction, while n2 and n^ may be complex.

Consider light incident at the boundary between the immersion medium

and film. The cosine of the refraction angle is

- (

^

^^""h )

1/2

(1)

The parallel and normal reflection coefficients for light incident at this

boundary are:

n2Cos(J)^ - n^cos(})2

and

= ——i^r:

-— (2) 12 n2Cos{})j^ + n^cos(p2

n^coscD^ - n^cos(D2 ^ 1 T 2— (3) 12 n^cos(})^ + n2Cos(j)2

-3-
-4-
P s respectively. The reflection coefficients, r^^ and r^^ the boundary

between the film and substrate are given by similar expressions.

D S The total reflection coefficients, R and R that include the

contributions of reflections from lower boundaries are given (15) by:

P P

r*^ + r^„ exp D

rP = _12 23

1 + rl^rl^ exp D

^ =

3 I (5)

1 ^12^23 ^'^P °

where cos values needed for these reflection coefficients are given

by an expression similar to eq (1) and D represents the quantity

and

D = -47Tin2Cos(t)2d2/X (6)

where X is the wavelength of the light used, in vacuum, and i = / -1

The ratio of the parallel and normal total reflection coefficients is

defined as p:

P = RP/R^ (7)

This may be expressed in terms of the relative attenuation and phase shift

of the parallel component with respect to the perpendicular component that

occurs, represented by the azimuthal angle A, and relative phase shift by;

p = tani|; exp (iA> (8)

Thus p is determined from ellipsometer readings.

The value of the complex index of a reflecting surface can be calculated

from the equation

n^ = n^tanc})^ 1 - 4psin (j)^

(p+1)^

1/2

(9)

where <p^ is the angle of incidence and p is determined from ellipsometry

measurements on the base substrate.

-5-
Further details of the technique are given in references 16-20 and

our application to the very analogous problem of the adsorption of synthetic

polymers from solution is given in references 21-25. In summary, for a bare

metallic surface two parameters are obtained from the instrument readings:

tan ip, the ratio of the magnitude of the reflection coefficient for light

polarized with its electric vector in the plane of incidence to that polarized

normal to the plan of incidence, and A, the relative phase difference for

these two polarizations. From these measured quantities it is possible to

calculate directly the optical constants of the surface. All other parameters

required for this calculation such as the wave length of light, refractive

index of immersion medium, etc., are obtained by independent means. If this

surface is now covered with a thin film, different values of ^ and A result.

From these new values and the previously determined values of the optical

constants of the surface, the thickness and refractive index of the film can

be calculated by the equations of Drude (15) . Although these equation are

not solvable in closed form, answers may be determined to any desired degree

of accuracy by iterative methods, necessitating the use of electronic computers

for most applications (26).

Shown in Figure 2 is a schematic diagram of the ellipsometer . Photographs

of the instrument are given in Figures 3 and 4. In normal use, the cell is

surrounded by a constant temperature jacket. A diagram of the cell is shown

in Figure 5.

The model treated in the Drude equation is that of a homogeneous film

of constant refractive index with discrete boundaries. However, an adsorbed

polymer film, consisting of a mixture of polymer and solvent, would be

expected to be an inhomogeneous film, with the poljnuer concentration and hence

refractive index decreasing with the distance from the surface. The use of

the Drude equations will result, therefore, in an "average" thickness value

and it is necessary to relate this calculated average from a homogeneous film

model to the inhomogeneous adsorbed polymer film (27), and if possible,

to molecular parameters.

Infrared Difference Spectroscopy

The interaction of the chromophores of an adsorbed molecule with a surface frequently results in a shift of their characteristic spectral

adsorption bands. Typically, for the protein studies presented here, a shift of -20 cm~l of the amide I band for free and bound carbonyl groups was

observed. (See Figure 6.) If these bands were well resolved, one could

immediately use the optical density of the shifted band to determine the

bound fraction, p, the fraction of the carbonyl chromophores of an adsorbed

protein molecule directly in contact with the surface. Knowledge of the

amino acid composition of the protein would then enable a calculation of

the average number of carbonyl attachments per molecule.

-6-
-7-

•H

00

cueo

COa-

•H

M-lo

a.

cd

>-i 00ou

o

42

u

3

bO

•H
-•10-
-11-
Usually, there is a significant overlap of the two bands

and difference techniques must be used. This is illustrated in Figure 7.

Fontana and Thomas (28) originally described a method whereby a polymer

is adsorbed from solution on a high surface-area powder, the suspension

centrifuged, and an infrared difference spectrum recorded for the resulting

gel. The signal due to the solvent and unbound chromophores of the adsorbed

polymer are compensated by adjusting the pathlength of the reference cell

containing polymer solution. This results in a resolved spectrum of the

adsorbed chromophores. This technique was modified (29) to permit a direct

analysis of the suspension, thereby removing uncertainties in polymer

concentration and has been used by a number of investigators (30-32) to study

the conformation of synthetic polymers adsorbed from organic solvents.

For the determination of the bound fraction for an adsorbed protein,

it is necessary to use^D^O solutions of deuterated proteins to obtain a window in the 1650 cm region. Although the shoulder of a DO band

exists just below this region, preventing the use of a variable pathlength reference cell, matched cells can be employed, and the following

analysis was used to resolve the difference spectrum arising from a protein solution-silica suspension in the sample beam versus the same protein solution in the reference beam (see Figure 7) . A difference spectrum

for bovine serum albumin is shown in Figure 8.

The absorbance at the frequency V° of the unshifted band is given

by Beer's law

A° = £°C^ L + e°C L + £°C^ L - £°C L (10)

f u,a a b r r

where e'^ and are the extinction coefficients of the unbound and bound

a o carbonyl groups at the frequency V , L and L are the path lengths of the

sample and reference cells, is the concenEration of unbound groups of

free protein in the suspension, C is the concentration of unbound groups

of adsorbed protein, is the concentration of bound groups, and C is

the concentration of carbonyl groups in the reference cell. With tfie total

concentration of groups C = Cf + -i- ^ and realizing that

e*-*Cj.Lj. = e L(C + C ) , where C^j is the change in concentration (dilution

effect) due to the addition of the non-porous powder, the bound fraction,

p, which is defined as C^/C where is the concentration of all groups

on adsorbed molecules (i.e., change m protein solution concentration as

a result of adsorption) , is given by

P

= A° + £°C L

a

C L(e° - e°) o

a a V

(11)

-12-
CO

LU

LlJ

CO<

UJ

LUO

LU

cr

LJ

Ll.

Ll Eo

CO Eo

CO

LO

CO

U-l I

LU CO

CO <=> ^ 1i

LLJ LLJ I od o

Li- CO

CO H-»

k LLJ ac: Q_

era

LlJ

CD CO LLJ CO

a::

c=> CO =s CO

£=) <:> ca

•<r CO 0: i 3»iaSQQ5QSBH5QSKBQSB

i«n9SflS»SW«SQSHS

CD CO I I I QC OJ

CO

cn 5 <=>

COI <=>Io

c_:>

4-1 c

(UB

01u3

CO

cc

(UE

(Uo

(U

>-l (U

H-l

M-l •HTJ

na

0)

n)

»^oo

4-1

(U

(U

00

-13-

To determine e'^ , the reference solution is diluted until the signal due

to unbound groups is nulled. This establishes the condition £ ^[-^j- ~

£ L(C^ + C ), which in conjunction with Eq. 10, yields

f u,a , 1 > /

£ L

a

where A*^' is the new absorbance at V . Elimination of C between Eqs. 10 and

Eq. 12, expressing the result in terms of known quantities, and solving

for e^, yields

a A°'£°

' £° = . (13)

A ' - A - £ LC,

d

Computer Simulation

The computer simulation studies of the adsorption of lysozyme utilized

the known atomic coordinates (33) determined from X-ray crystallographic

studies. For convenience in these calculations, a coordinate system with

the X axis passing through the amino acid residues glycine 126 and proline

70, and Y axis passing through glycine 126 and alanine 42, was chosen, as shown in Figure 9. The process of adsorption of a given orientation of

lysozyme onto a planar surface was simulated as follows. For a given

orientation of the lysozyme molecule with respect to the surface, the molecule

was moved toward the surface until the first contact was established between

an atom of the molecule and the surface. The molecule and surface were then

held fixed. The question was then asked: At what distance normal to the

surface should a hypothetical plane parallel to the surface be placed such

that a given number of carbonyl groups are located between the plane and

the surface? This distance was then determined, the molecule removed from

the surf ace^ rotated, and the process repeated. All possible orientations

of lysozyme with respect to the surface resulting from rotation in 5* increments

about the X and Y axes were tested. The vicinity of the minimum distance

determined by this procedure was then probed in 1" increments.

Radiotracer

As the adsorption of proteins from plasma occurs from a complex mixture,

their rates of competitive adsorption are fundamental in determining the

eventual state of any given protein. Such rates must be accurately determined

on the single proteins before the effects of competitive adsorption can be

assessed. Radiotracers are convenient for the accurate measurement of amounts

of adsorbed protein. The adsorbing solution concentrations can be varied

over a wide range and adsorption times on the order of 5-10 seconds can

be studied. Furthermore, by the proper selection of tagged components, the

relative amounts adsorbed of two or more proteins can be determined even when adsorbed simultaneously from the same solution. We have previously

applied radiotracer techniques to studies of the adsorption of labeled

synthetic polymers utilizing both tritium and -^^C.

-15-
llJ NO

CO

I-o< GO

•H

4-t iH

(J

C

y

to iH

u

tX 0) C 3 O (X 'O o

(U

u o o

CO iH

CO "xi(U

0) CO O 0)

CO 4-1 CO VH >^

CO o cu

4-1

C CO o c

•H -H

+J 13

CO

U

4-t

O c o

(U o

CO

CU bO M C a 'H

t-1 o O CO •H

4-1 (U CO O S CO (U M-l

>-l

O 3

CO CO

as

•H

[±4

-16-
A major problem in the investigation of protein adsorption by

radiotracer techniques is the removal of the sample from the adsorption

solution without transferring any of the protein from the air-solution

interface. It is also necessary to wash away the "carry-out" solution

without desorbing any of the adsorbed protein. However, a technique

that includes washing, no matter how short the washing period, may remove

an unknown amount of adsorbed polymer. Comparison of the adsorbed

quantities with those obtained by other techniques, such as ellipsometry

,

however, permits an evaluation of the quantities that we removed by a washing process.

-17-
EXPERIMENTAL

E llipsometry ^

The following materials were used for the ellipsometric studies.

Human serum albumin (4x crystallized) , bovine prothrombin (Cohn III-2) , ^ and e-aminocaproic acid were obtained from Nutritional Biochemicals (NBC)

and used without further treatment. Human fibrinogen (NBC) was purified

by the procedure of Batt, et.al. (34), a modification of a procedure due

to Laki (35)

Commercial ferrotype plate consisted of chromium electroplated on either brass or stainless steel. Platinum was of 99.95% purity and rolled

to a mirror finish by Engelhard Industries. Quartz was fused silica, no. 7940,

from Corning Glass. These materials were cleaned before use with hot 50:50

HN03:H2S04 followed by three rinses in boiling distilled water. They were

then heated for five minutes at 500° in a muffle furnace and placed while

still warm into the adsorption cell filled with buffer.

Low temperature laminar (LTL) type pyrolytic graphite was kindly

supplied by Dr. Jack Bokros of General Atomic Corp. Commercial polyethylene

sheet was cleaned in ethanol and cast under vacuum at 180° between plate glass

sheets. Several samples were prepared from NBS Standard Reference Material

1475 linear polyethylene which has a density of 0.978 g/cm-^» also by casting

against glass under vacuum. All results on these two types of polyethylene

were identical. The carbon and polyethylene samples were cleaned before

use by washing with ethanol for two weeks in a Soxhlet extractor followed

by a similar washing with distilled water.

All solutions were made up in pH 7.4 phosphate buffer and concentrations

were determined by UV spectroscopy. All adsorption measurements v^ere made

in a ceil thermostatted at 37°C.

*Certain commercial materials and instruments are identified here

and elsewhere in this publication in order to adequately specify the

experimental procedure. In no case does such identification imply

recommendation or endorsement by the National Bureau of Standards, nor does

it imply that the equipment or instruments identified are necessarily the

best available for the purpose.
Each adsorption run consisted of first determining the optical constants

of the bare surface while under the buffer solvent. Buffer was then withdrawn with a syringe and the cell refilled with additional buffer and the

optical constants redetermined. Solvent exchange was repeated until no change in optical constants was observed. If changes were observed after

three such exchanges, the surface was discarded. The solvent was then

exchanged for solution and measurements were taken as a function of

adsorption time. Due to refractive index inhomogeneities in the adsorption

cell, stable readings cannot generally be made in less than ten minutes

following any exchange of solvent or solution. Blank runs made with surfaces

kept under buffer for several days established the amount of drift in ellipsometer

readings to be expected for these time periods. In general, drift was negligible (< 0.05°) for times up to 24 hours. Longer times occasionally

led to substantial changes.

Infrared Difference Spectrometry

Bovine serum albumin (4x crystallized) , bovine fibrinogen (Cohen fraction

I, 60% clottable) , bovine prothrombin (fraction III-2) , and lysozyme (chicken

egg white, 3x crystallized) were obtained from Nutritional Biochemicals

The serum albumin and lysozyme were deuterated (36), lyophilized, and stored

in vacuo at A'C. The prothrombin was dissolved in O.IM D2O phosphate buffer

pD 7.4, dialyzed overnight against this buffer, and filtered through a well

washed 0.8 ym pore size filter just prior to use. Fibrinogen was purified

by the method of Laki (35) with the following modifications: The ammonium

sulfate precipitate was resuspended in the pD 7.4 phosphate buffer and

dialyzed overnight aginst the same buffer. The resulting solution was filtered

and used immediately.

Poly-L- lysine and poly-L-glutamic acid of average molecular weights

139,000 and 102,700, respectively, were obtained from Nutritional Biochemicals.

The polyamino acids were deuterated prior to use by dialyzing overnight against

D20-phosphate buffer. The a-helix and random coil forms of poly-L-lysine

were obtained by adjusting (37) a portion of the deuterated material to pD 11.5

and 4.8 with Del and NaOD, while the helix and coil forms of poly-L-glutamic

acid were generated by adjusting (38) the pD to 5.4 and 11.4. Polyamino

acid concentration changes were measured spectrophotometrically (39) using the

optical density difference at 215 and 225 nm.

The mild heat denaturation of serum albumin was carried out at 66°C (40)

The quantitative, limited, reduction of disulfide bonds of serum albumin was prepared by the method of Sela et. al . (41) with the omission of urea and a reaction time of 1.5 hours.

The cross-linking of serum albumin was accomplished using diethyl

malonimidate dihydrochloride prepared from malononitrile and HCl gas in

anhydrous ethanol-dioxane according to McElvain and Schroeder (42). The

reaction with serum albumin generally followed Dutton et. al. (43) , using

a 1.5% protein solution with reaction for 1 hour.

-19-
The O.IM phosphate buffer was prepared using 99.8% D2O and

deuterated salts. Measurements of pD were standardized against NBS

standard reference materials 2186-1 and 2186-11. The pD of solutions

was adjusted with concentrated DCl and NaOD. Ionic strength adjustments

were made with KCl.

The adsorbent used for all experiments was a fumed, non-porous

silica with a nominal particle size of 0.012 ym (Cab-O-Sil M-5, Cabot

Corp.) The adsorbent was heated in vacuo at 110°C overnight and stored

over silica gel just prior to use. The surface area, as determined by

BET N2 analysis, was found to be 204 ± 20 m^/gm.

Data for adsorption isotherms were obtained at 23.5 ± O.S^C by shaking

known amounts of silica and protein solution for three hours (judged to

be equilibrium from rate studies showing no further increase in adsorbance)

The silica was then removed by filtration. The amount of adsorbed protein

was determined from the protein concentration changes measured spectrophotometrically at 280 nm using accepted extinction coefficients for

fibrinogen and prothrombin (44), serum albumin (45), and lysozyme (46). To

check for contamination, the ratio of silica to solution volume was varied

with no effect on the protein adsorption isotherms. For studies of

adsorbance as a function of time, the quantities of silica and protein

solution were suitably scaled up and at various times aliquots withdrawn

and filtered.

For bound fraction studies, 0.029gm silica per ml protein solution

was found generally to give stable suspensions suitable for infrared

analysis. The difference spectrum of the protein-silica suspension versus

the same protein solution was obtained from the region 1750-1550 cm"-*- using

matched 0.1 mm CaF2 cells at lOx ordinate expansion. The extinction

coefficient, e^, was obtained by scanning successive dilutions of the

reference solution against the protein-silica suspension and noting the

dilution at which the peak due to the unbound chromophores diappeared. Each

reported bound fraction value represents the average of from two to four

infrared scans on the same protein suspension.

While contact of the D2O solutions with the atmosphere can lead to the

incorporation of H2O, the rapid conversion H2O + D2O ^ 2H0D insured a window

in the 1650 cm"-'- region. The resulting HOD, however, does obscure the 1500

cm"! region where the amide II band involving N-H occurs. Therefore, only

contacts of the carbonyl group were measured and used to calculate the

bound fractions reported in this paper.

Radiotracer

The radiotracer experiments utilized the same chrome and platinum

as used for the ellipsometry studies. '-^-'I labeled human albumin was

obtained from Squibb and quartz from General Electric (Type 151).

The chrome slides were cleaned in hot benzene followed by 10 minutes of

heating in a muffle furnace at 500°C. The quartz and platinum were cleaned

with hot 1:2 HNO„:H SO, for one hour followed by heat treatment at 500''C

-20-
for 10 minutes. The slides were placed in buffer while still hot and

were never subsequently exposed to air.

14 14

C hexadecane and C stearic acid used for surface roughness

determinations were obtained from New England Nuclear Corp.

Solutions of the unlabeled protein were made up by weight in pH 7.4

phosphate buffer and 131i labeled protein solution was added by syringe.

Adsorption was carried out in a covered glass vessel equipped with a magnetic stirrer and wrapped with heating coils to maintain a temperature

of 37°C.

In order to establish the specific activity of the labeled protein

solutions, 1 to 10 A aliquots of the solution were taken and evaporated

to dryness on steel planchets. These were counted at the same time as the

adsorption samples to minimize decay corrections. Measured activities

could be made independent of sample size by coincidence time corrections

but these were usually high for the large aliquots. Separate experiments

established that the quartz and chrome samples had the same backscatter as the steel while the platinum showed 25% more backscatter and the activities

were adjusted for this.

All counting was done in a Nuclear Chicago 1105 low background beta

counter with a mylar window operating in the G-M mode. The adsorption

slides were always masked with a lead sheet containing a circular hole in

order to maintain consistent counting geometry, define the counted area,

and to eliminate edge effects.

-21-
MOLECULAR EXTENSION

Time Dependent Behavior

Two aspects of the ellipsometric results should be considered: a) the

time dependence of the adsorption process and b) the long time or equilibrium

values. For example. Figures 10 and 11 show the time dependence of the

thickness or molecular extension and adsorbance, respectively, of fibrinogen

adsorbed on chromium. The error bars in Figure 10 are calculated maximum

uncertainties based upon estimates of instrumental precision. The corresponding

error limits in Figure 11 are smaller than the sizes of the points. While

no change is seen in the extension during the time period shown, there is a

sma].l but definite increase in adsorbance. Rates should not be inferred from

these results as the ellipsometer cell was not stirred for these experiments.

Although duplicate runs, in general, may give somewhat different values of

extension and adsorbance, the general features of increasing adsorbance

with constant extension are always observed within an individual set of

data for this protein and surface. , Extension measurements made at adsorption

times shorter than 20 minutes generally show greater scatter, but they are generally randomly displaced from the average value.

The constant value of extension with time can be interpreted as a lack

of significant molecular rearrangement of the protein molecule once adsorption has taken place. In the early time periods, additional molecules

are still arriving at the surface as evidenced by the increasing adsorbance,

but the surface pressure of these molecules is not sufficient to cause the

molecules already adsorbed to change their conformation. Nor, apparently,

do the late arrivals adopt a st gnif icant 1 y more extended conformation than

the majority of the adsorbed molecules. It is recognized that conformational

changes may occur in the first few seconds or minutes of adsorption. However,

it should be noted that a random coil homopoljnner has been shown (22) to

undergo a significant change in extension during the time periods accessible

by this technique, indicating more conformational freedom than that shown by

the proteins studied here.

The absence of change in extension with adsorption time is a general

feature found for all three proteins on all of the surfaces examined to

date. Further examples can be seen in the data for the adsorption of prothrombin on chromium (Figures 12 and 13) , laminar carbon (Figures 14 and 15)

,

and polyethylene (Figures 16 and 17). The differences in the error limits

of adsorbance seen in these figures is due to the low refractive index of

polyethylene causing a loss of sensitivity.

An increase in adsorbance was not always observed. An example of this

is shown in Figure 18 which gives the adsorbance of albumin on chrome where

no discernable trend can be seen. Under certain conditions, a decrease in

adsorbance with time has been noted when unpurified Cohn Fraction I fibrinogen was adsorbed. The adsorption of Cohn Fraction I on the high

surface energy metals and quartz was qualitatively the same as the purified

-22-
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-31-
fibrinogen although at long times txtension and adsorbance values did

not agree, within the error limits, for the two materials. On the lower

energy carbon surface the adsorbance increased to a maximum and then

decreased steadily as shown in Figures 19 and 20. An even more rapid

decrease is seen on polyethylene (Figures 21 and 22) although readings

after 400 minutes were imprecise because of the small amount of material

left on the surface.

It can be speculated that this removal may be due to the action of plasmin,

a fibrinolytic enzyme, which could come from the activation of plasminogen

which is presumably a contaminant of Cohn Fraction I (47) . Plasminogen

contamination was surmised on the basis of short clot lysis tiroes from

the Fraction I fibrinogen. This explanation was tested in three ways.

First, f ibrinolysin, a factor elaborated by streptococcal bacteria which

activates plasminogen, was added to the unpurified Fraction I and as shown in Figure 23, the rate of removal was increased. Secondly, e- aminocaproic acid was added and was shown to retard the rate of removal

(Figure 24). e-Aminocaproic acid is an inhibitor of the plasminogen to

plasmin interconversion and also a mild inhibitor of plasmin action (48).

The Cohn Fraction I was purified by gel permeation chromatography under

conditions reported to remove plasminogen (49) and the decrease was eliminated (open symbols in Figure 25) . Fibrinogen purified by ammonium

sulfate precipitation shows the same adsorbance behavior as the chromatographed fibrinogen but the clot lysis times are not as long. Finally,

addition of fibrinolysin plus a small amount of unpurified fibrinogen to

the purified fibrinogen restores the decrease (closed symbols in Figure 25)

This evidence is not conclusive, but it is highly suggestive that the

plasminogen-plasmin system is responsible for the removal of fibrinogen from

the surface. It is interesting to note that the action of plasmin appears

to be rather specific for the adsorbed fibrinogen as the supernatant solution

remaining after almost all the fibrinogen has been removed from the surface

still contains enough fibrinogen to be clotted by thrombin.

The reason for the lack of removal on the higher energy surfaces is

not clear. It can be speculated that the plasmin is tightly adsorbed to

the surface in these cases and therefore inactivated or that the fibrinogen

is held in a conformation which hinders enzymatic attack. It may even be

that proteolysis does indeed occur, but that the fragments are. also held

in these cases and no change in adsorbance is seen. We have no further

evidence that bears directly on any of these contentions.

Equilibrium values

The equilibrium adsorbance and extension values for the proteins and

surfaces studied are summarized in Table I. It should be emphasized that

the different proteins were adsorbed at concentrations that approximate

physiologic ones and are therefore quite different from one another. Albumin

was adsorbed at 30 mg/ml, fibrinogen at 2.5 mg/ml and prothrombin at 0.2 mg/ml.

The high albumin concentration gave a solution with a refractive index close

-32-
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-39-
to that of the adsorbed film. When this was coupled with a low substrate

index, the resulting lack of sensitivity gave exceedingly wide error limits

and meaningful measurements were not possible. This condition accounts

for the blanks in Table I. Measurements can be made at much lower

albumin concentrations, but they would then not be comparable to those on

the high energy surfaces.

Two main features should be noted in the table. First, in spite of

the large concentration differences in the proteins and the varied nature

of the surfaces, the adsorbances cover only a rather small range. There

is also no trend in adsorbance with surface energy. If surface energy is

estimated by the critical surface tension for wetting, then the surface

energies range from high to low in the series platinum, chrome, quartz,

carbon, and polyethylene. There is, however, a correlation of surface

energy with extension. Such an increase in extension with decreasing surface

energy would be in accord with flat, tightly held conformations on the

higher energy surfaces and looser, more extended conformations on the lower

energy surfaces.

The adsorbances in Table I are all consistent with monolayers of

protein of unperturbed molecular dimensions, but the concept of a monolayer

may not be applicable to such large molecules.

Finally, the extensions given should not be construed as molecular

dimensions of the adsorbed protein. The model assumed for the calculation

of this extension is that of a film of homogeneous refractive index with

discrete boundaries. Obviously, any large adsorbed molecule will show

some refractive index inhomogeneities normal to the surface and this will

affect the calculated extensions directly. This problem has been considered

by McCrackin and Colson (27) who have shown that extensions calculated

assuming a homogeneous film may be converted to root-mean-square segment to

surface distances if the actual refractive index distribution is known. For

example, the homogeneous film thicknesses have to be divided by factors of

from 1.5 to 1.7 if the refractive index distributions are linear, gaussian,

or exponential. As we have no estimate of the refractive index distribution

of an adsorbed protein we have no way of making such a conversion for this

data but the homogeneous extensions may perhaps be considered as upper limit

approximations to molecular dimensions. The relative values of the extensions

on the different surfaces may properly be compared assuming that the

refractive index distribution does not change markedly from one surface to

another.

-40-
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-41-
RESULTS AND DISCUSSION

BOUND FRACTION

Equilibrium Conformation Studies ^

E{i{)(lcX 0{j SuAj^ace PopLLtaJxon - The isotherms of serum albumin,

prothrombin, and fibrinogen are given in Figures 26-28. Figures 26 and

27 also include values of the bound fraction for serum albumin and prothrombin, respectively. Portions of the fibrinogen isotherm were inaccessible

for bound fraction measurements using the technique described above because

of the large adsorbance of this protein (initial concentrations were limited

for fibrinogen because of the low solubility of fibrinogen in the D2O - phosphate buffer).

For serum albumin and prothrombin, no systematic change in the bound

fraction was apparent over the measured portion of each isotherm. The

mean values with their computed standard deviations were 0.11 ± 0.02 and

0.11 ± 0.03 for serum albumin and prothrombin respectively. Results for

fibrinogen showed an increase in the bound fraction with increasing amount

adsorbed. In Figure 29 are shown the results of a set of four experiments

carried out for the accessible portion of the isotherm. The dashed line

represents a least-squares linear fit to the data. The unstable nature of

the f ibrinogen-silica suspension is at least partially responsible for the

large scatter in the values of the bound fraction, p, and, in conjunction with

the high adsorbance, prevented measurements of p at larger adsorbance values.

Using the known amino acid compositions of serum albumin, prothrombin,

and fibrinogen, the bound fractions reported translate into a minimum of 77

contacts with the silica surface or serum albumin, 80 for prothrombin, and

176-703 for fibrinogen, as shown in Table II. If the adsorbed protein

molecules were spread nearly flat on the surface, large values of the bound

fraction would be expected. Adsorption of poly (ethylene o-phthalate) (31)

,

poly(methyl methacrylate) (30), polystyrene (32), and poly(A-vinyl pyridine)

(29) on Cab-O-Sil gave bound fractions of 0.37, 0.35, 0.24, and 0.35

respectively. In each of these cases, the synthetic molecules appear to lie

close to the surface in a presumably spread condition. Since bound fractions

of 0.11 were obtained for prothrombin and serum albumin, the adsorbed protein

molecules are still relatively unchanged by the surface, despite the large

number of attachments and great interaction.

The bound fraction data can yield information on changes in the

conformation of the adsorbed protein molecule as a result of increasing

competition for surface sites. A decrease in the bound fraction values

with increasing population of protein atoms at the surface would be expected

if changes in conformation were to occur. Such changes in conformation of

synthetic random coil polymers have been observed (29,30,22). It has been

suggested (50,51) that an adsorbed serum albumin molecule should initially

unfold increasing the value of p, which would presumably then decrease with

increasing surface population. Our results for serum albumin and prothrombin,

Figures 26 and 27, show that the bound fraction remains constant, indicating

that the conformation does not change as the surface population increases.

The relatively low value of p for these proteins as compared to synthetic

random coil polymers, and the constant value of p over the isotherms, strongly

suggests that the internal bonding and disulfide cross-links of the globular

proteins are sufficient to preserve the basic tertiary structure of the molecules.

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CM• 1—1• oa o« o O o o o o o d NOiiovyj ONdoa

»A6-
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•H

0) 4JOuuo

<4-l

(« wQ

(3o

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CO H

CO

4-1

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CO •Ul - o O

0) 0) H CJ d CO

0) V4 H 3 O CO

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C3 -a o O O M CO

>-i

0)oc • CO a ,Q 0 }-i o O r-i CJ u (0 0 o ^3

<: t>0 rH e

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cr 1

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CO 0 CO 0 00

•H ^ 00 iH a i-i to • • CO O 0 o CM o ds

^4

(U 0 O 3 0 0 0 CO bO

•H ^ X M 00 O 0

CO <

0)o o00 o o o

00

iH

O 0303

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jjo

cnoI OCM

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000

0)

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-47-
The adsorption behavior of fibrinogen (Fig. 29), which shows a direct

dependence of bound fraction on adsorbance, does not necessarily represent

a conformational change. As noted above, previous experience with synthetic

polymers has shown that conformation changes resulting from an increase in

surface population are accompanied by decreases in the bound fraction. oThe

bovine fibrinogen molecule is rodlike with a length of between 400-600 A (52)

and can easily interact with more than one silica particle. It has been

postulated (53) that the solubility of fibrinogen is due to electrostatic

repulsion between regions of high negative charge density localized in

f ibrinopeptides A and B. If adsorption and interaction with the hydrophilic

surface were to effectively neutralize this repulsion, fibrinogen molecules

adsorbed to different silica particles could interact like fibrin, thereby

increasing the surface attachments of each fibrinogen molecule. Such an interfacial aggregation (51) could result in an increase in bound fraction

values with increasing adsorbance, without significantly hindering further

adsorption to the remaining available surface.

Our data for maximum adsorbance can be converted to amounts/unit area using the measured surface area. (Surface area determinations of Cab-O-Sil

using stearic acid adsorption (54) agree with the BET-N2 analysis). These

results are presented in Table II and agree quite favorably with those found

for adsorption of human blood proteins on quartz using ellipsometric techniques

as shown in Table I. Results obtained by MacRitchie (51) for adsorption of

bovine serum albumin on a silica surface (also 200 m /gm) at pH 7.5 were only

one third as great as that found in the present study. The reason for this

difference is not obvious.

UaAiatLon 0^ pV and Ionic StAzngth - Experiments were conducted to

determine the effect of pD and ionic strength on the adsorbance and bound

fraction of serum albumin. The results obtained for an initial concentration

of 4.3 mg/ml serum albumin at ionic strengths of 0.1, 0.2, and 0.5 in D2Ophosphate buffer are presented in Figure 30. The curve drawn represents the

effect of pD on adsorbance for ionic strength 0.1. The figures given along

the curve are the values of the bound fraction at that pD. Since no effect

of ionic strength was observed on the bound fraction within experimental

error, the values of p are the average for the three ionic strengths. Both

the adsorbance and bound fraction reach a broad maximum at approximately pD

5.2 for ionic strengths of 0.1 and 0.2. For these two values of ionic strength,

there is no effect on the adsorbance at or near the maximum, while strong

effects are noted at the extremes of pD. The pD profile of adsorbance for

I = 0.5 shows no maximum, although there appears to be an asymptote in the

acid region. A maximum in the bound value also occurs near pD 5.2, although

the profile is different from that of the adsorbance vs pD curve. While the

adsorbance values at pD 7.4 are near the plateau values, those at pD 5.0 are

only on the knee of the isotherm (plateau adsorbance at pD 5: A = 308 mg/gm

C^q^il =3.0 mg/ml).

The adsorbance-pD profiles of serum albumin on silica are similar to those

observed by Bull (55) on glass. Silica exhibits a decreasing negative charge over the pH region of 8.0 to 2.0 as determined from electrophoretic mobility

(56,57) and sedimentation (58) methods. If electrostatic attractions between

the adsorbing protein and silica were the factor governing adsorption, the

adsorbance should increase as the pD decreases, since the isoelectric point

of serum albumin occurs at pD 5.0. The conformation of bovine serum albumin

in solution undergoes interesting changes with pH (59). At values acid to the

isoelectric point, the molecule reversibly unfolds and becomes flexible, until

48-
;ure 30: Adsorbance vs

pD for bovine serum albumin at 0.1 (CJ), 0.2 (A),

and 0.5 (^) ionic strength on silica. The curve represents ionic

strength 0.1. The values given on the curve represent the bound

fraction at a given pD. These values were the same for all three

ionic strengths. -49-
finally at pH 2.5 - 4.0 with I > 0.1, an aggregation occurs (60). The

experimentally determined bound fraction (Fig. 30) as a function of pD is,

therefore, the result of a number of contributing effects which cannot now ^ be assessed. The coincidence of the maxima near pD 5 of the adsorbance and

bound fraction profiles strongly suggests, however, that interactions between

the adsorbed protein molecules are important in determining both the adsorbance

and conformation. This is consistent with the results obtained in general

for adsorbing polyelectroly tes (61)

Relating the plateau values of protein isotherms for a solid-liquid

system with a close-packed monolayer can be very misleading. Using accepted

figures for the molecular geometry of serum albumin in solution (62) , one would calculate an adsorbance for our system of 520-1460 mg/gm depending on whether the molecule is adsorbed side-on or end-on. We do not approach

these values under any conditions. At pD 3.35, the plateau adsorbances for

ionic strengths of 0.1, 0.2, and 0.5 are 142, 193, and -365 mg/gm, respectively.

The bound fraction (conformation) is, however, not a function of the ionic

strength (Fig. 30). One would, therefore, predict identical plateau adsorbance

values for each ionic strength. It is clear that caution must be exercised in

interpreting the available surface area per adsorbed molecule as an indication

of changes in conformation upon adsorption.

Non-Equilibrium Conformation Studies

Pependence o^j CoYi{^omatLon. on kcUonptlon Time, - The possibility that the

conformation of the adsorbed protein might vary as a function of the length of

time for adsorption was tested by determining the bound fraction for each

protein periodically following the formation of a suspension. Figure 31

illustrates some typical results obtained for serum albumin, prothrombin, and

fibrinogen where the bound fraction values are listed over each curve at the

time of measurement. Although the rates of adsorption cannot be compared since

the initial conditions are different for each experiment, it is clear that there

is no variation in the bound fraction for any of the proteins over the time

period investigated at these conditions.

Our studies of the effect of length of time for adsorption on the bound

fraction indicate that molecular rearrangements do not occur following adsorption

at the concentrations studied. Although the adsorption rates for prothrombin

and fibrinogen were extremely high as a result of the conditions required for

bound fraction measurements, the relatively slow adsorption of serum albumin

allows measurements of p as a function of time which shows that the conformation

is not dependent upon the surface population, as was found in the equilibrium

adsorption studies (Figs. 26 and 27). The slight decrease in adsorbance of

prothrombin with time shown in Fig. 30 is possibly caused by the dissociation

of an aggregate at the low final solution concentration. Prothrombin has been

reported (63) to associate reversibly at low ionic strengths.

CompaAjOtyivQ. Rcutu^ AcUoh.ptA.on - To compare the rates of adsorption of serum albumin, prothrombin, and fibrinogen, each was adsorbed at an initial

concentration of 1.75 x 10"^ M with 0.004 gm silica per ml protein solution.

The rates of adsorption are quite different, as shown in Figure 32. These

-50-
CD

CP

LUO

<

m

cro

(/) Q

<

256-

252

248-

.11. .12 — .12.

Prothrombin, C=0.24

206-

202-

^l_2 .18_.24 .25 .33 13 &

Fibrinogen, C=0.069

132-

128-

124-

120

116

.15 .16 A .12

Serum Albumin, C=0.98

25 50 75 100 125 150 175 200 225

TIME, min

Figure 31: Adsorbance vs

time for bovine serum albumin, prothrombin, and

fibrinogen. Values of bound fraction are given over each curve

at the time measured. Adsorption on silica at pD 7.4.

-51-
1050

25-

°

0 10 20 30 40 50 60 70 80 90

TIME, min

Figure 32: Adsorbance vs^ time for 1.75 x 10~ M bovine serum albumin,

prothrombin, and fibrinogen. Adsorption on silica (0.004 gm/ml

solution) at pD 7.4.

-52-
differences are more apparent when the data is presented as the ratio of

the adsorbance at a given time to the value at the plateau, as is shown

in Table III. Clearly prothrombin is more rapidly adsorbed for all time

intervals. Serum albumin and fibrinogen, while very similar in their rates

at early times, differ markedly in their approach to equilibrium.

The findings of the comparative rate study for the three proteins on silica

are very similar to the rates observed by Lyman (64) for adsorption of the same proteins on flat polymeric sheets. To determine if the adsorption process

is diffusion controlled, the results we obtained for fibrinogen were subjected

to an analysis described by Crank (65) for adsorption on spheres from a solution

of finite volume and known initial concentration. Using the nominal diameter of

120 A of Cab-O-Sil as the particle size and a diffusion coefficient of 2.02 x

10" 7 cm^/sec for fibrinogen, one calculates that adsorption should be complete

in 5 X 10"^ sec. It is apparent that factors other than diffusion must govern

the adsorption of the proteins studied on a finely divided silica.

Changes in Conformation of Serum Albumin Upon Adsorption

The conformational changes of blood proteins upon adsorption are extremely

important to any understanding of surface induced coagulation. A priori , one

does not know how much denaturation , if any, occurs upon adsorption of native

proteins. The effects of any such denaturation are necessarily included in

our values of the bound fraction. The presence of an infrared shift in the

difference spectrum for the bound carbonyl group does not necessarily mean

that protein denaturation has occurred upon adsorption. The shift may occur

for adsorbed protein, even if the tertiary structure has not changed. It is

possible, however, with the aid of certain assumptions to gain some insight

regarding the amount of distortion of the molecule that occurs upon adsorption.

The observed bound fraction for serum albumin can be considered to be the sum of two contributions: the first due to contacts permitted by the native

tertiary structure with the surface and the second resulting from surface

induced rearrangements that occur as a consequence of adsorption. These

contributions can be separated by determining the bound fraction of a highly

cross-linked, and therefore rigid, material. Cross-linking of serum albumin

with diethyl malonimidate dihydrochloride (43) produces a protein which is

resistant to denaturation, has the same electrophoretic mobility, and most

important, retains the same antigenic activity as native serum albumin. We

prepared such a cross-linked material which had 45% of its free amino groups

blocked (66). The average result of six experiments show in Table IV gave a bound fraction which was experimentally indistinguishable from that of the

native protein. We conclude that surface induced rearrangements, if present

in native serum albumin, are small.

As a means of assessing the extent of denaturation of native material

upon adsorption, we have denatured serum albumin with known, mild techniques

and determined the bound fraction of the resulting material. Table IV lists

the results for heat denaturation at 66*'C and thioglycollic acid reduction.

It is clear that the bound fraction for a given protein is strongly dependent

-53-
(1) UoW<

•H

0) 4JO

>-i o

•H

4-) O

CO

P4

CO H o e e CO •H

00

00

00§o ooc

•H

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siCO

54-
o

c

oo

U

3

O

43

cd H

1

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<u w

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0)

13O

-o o

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M

o

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U iH a> 0 o 4-1 fS 1 u vO O H

o CO aj VO o COo

u

Helie 1 CM

a;

-55-

<•
on its tertiary structure. Heat, which is known to cause the irreversible

aggregation of serum albumin (67) , leads to a bound fraction one third that

of native material. Mild reduction, where one disulfide bond was broken as determined by a p-chloromercuribenzoate titration (41) , leads to a bound ^ fraction of about 1-1/2 times that for native protein.

These studies also shed light on the conformation of native adsorbed

serum albumin. If the native protein were unfolded by an amount equivalent

to the breaking of one disulfide bond, an increase of 55 contacts (Tables IV

and II) would result. On the other hand, pretreatment of the serum albumin

in a manner which has been reported (67) to result in aggregates resulted in

a decrease of 50 surface contacts, implying that no aggregation occurs upon

adsorption of the native serum albumin. These results for denatured serum albumin show that bound fraction is sensitive to what in our view are small

conformational changes.

Conformation Studies of Model Compounds

ExpeAA.m2.yitcLt and Computer SAjmiiZjoutLon ol Ly^ozymz kcUiOKption - In order

to understand better the extent of the changes in protein structure which may

occur upon adsorption, computer simulation and experimental data were compared

for a protein of known structure, lysozyme. Accordingly, bound fraction

measurements were made at two different initial concentrations of lysozyme^^

where the ratio of lysozyme molecules to silica particles (assuming a 120 A

diameter) was 5.5 and 14.2. The experimental value of p = 0.12 thus determined

is equivalent to 19 carbonyl groups using the known amino acid composition of

lysozyme (68). The algorithm described in the technique section was then

applied to the known coordinates, and the minimum distance for 19 carbonyl

oxygen atoms from the adsorbing interface determined by inspection of the output for all possible orientations. With respect to the coordinate system

established, this distance was 3.00 A and occurred at angles of rotation of

124° and 0° about the X and Y-axes respectively. Figure 33 gives the distribution of carbonyl oxygen atoms near the surface for this particular adsorbed

orientation. Whjle the minimum distance for 19 carbonyl oxygen atoms is

approximately 3 A froip the interface, the mean distance assuming random

orientation is 7.62 A.

These results can be used as a model to gain some insight into conformational

changes accompanying adsorption. For the interaction distance of 1.88 A [the

N-H-N hydrogen bond distance for nucleic acids minus the N-H bond length (69) ]

,

only 13 carbonyl oxygen atoms compared with the 19 determined experimentally

are found to be interacting with the surface (Fig. 33), indicating a small

(with respect to changes in the radius of gyration) rearrangement upon

adsorption. One could also attempt to use a two state model and treat the

observed bound fraction as a linear combination of some fraction of totally

native and denatured material. (Bound fraction parameters describing the

native and denatured states could come from the computer simulation and

synthetic polymer studies, respectively.) Such an approach is limited, however,

since our work with serum albumin shows that the parameters would not generally hold.

-56-
i

LO

TCO LO C\J

1^

cn

LO

« CO

o

CO

o<

LO

' LU

CM ^

C\J

LO

<

I—

CO

LO

CD CD

(Uo

00 CO c

O iH CO

u-i o x; O -H W

3 U-l

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O CO

>-l O <U •H ^

a o

}-i CO CO O X) Ou

CO CO X)

CO Q)

(U 4.1

4J

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iw CO O 4-1 CO C -H O X)

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6 CO •H E

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u

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a. 00 o X xi D O 4-J

0-) CO

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00

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-57-
Potyamlno AcA.d Studies - Attempts to determine the bound fraction of

a polyamino acid of a known conformation were only partially successful.

\Vhile the a-helical form of poly-L- lysine (at pD 11.5) and poly-L-glutamic

acid (at pD 5.4) adsorbed to yield bound fractions of 0.60 and 0.50 ^ respectively, the random coil form of each polymer did not adsorb or give

a difference spectrum. This negative finding may be due to strong polymerpolymer interactions since both polyamino acids are highly charged in

their random coil form. The large bound fractions for the a-helical

forms is consistent with a bridging mechanism for adsorption. Assuming

3.60 residues per turn of the helix with a rise of 5.38 A, the length of

the poly-L-lysine and poly-L-glutamic acid were 1618 and 1180A, respectively.

Studies of polyamino adsorption, with lower molecular weights to avoid

bridging, on larger particle size polystyrene latex, to reduce polymerpolymer interactions, are planned.

-58-
RESULTS AND DISCUSSION

EFFECT OF ADSORPTION ON THE THROMBIN-FIBRINOGEN REACTION

One method of assessing the effect of adsorption on the thrombinfibrinogen reaction is to determine the kinetics of f ibrinopeptide release

for solution compared to adsorbed fibrinogen. Using Finlayson's techniques

(20), and using a Sakaguchi assay, we have obtained results for the control

experiment where all proteins are in solution. Experiments using fibrinogen

insolubilized on Cab-O-Sil have shown no measurable f ibrinopeptide release

and greatly increased clotting times. Tests have shown that the thrombin

in this system is being adsorbed and inactivated. This work will be pursued

using a different surface, detergent micelles, which have been shown to

enhance the activity of lysozyme.

COMPETITIVE ADSORPTION WITH UNLABELED PROTEINS

Work has been initiated to determine the competitive interactions

between blood proteins during the processes of adsorption, displacement,

and desorption. By taking advantage of the unique biological properties

of fibrinogen and prothrombin, one can study all pairwise combinations of

fibrinogen (coagulation with thrombin), prothrombin (isoelectric precipitation

at pH 5.3), and serum albumin without introducing any labeling atoms or groups.

The preliminary results obtained thus far show that fibrinogen interacts more strongly than serum albumin with a silica surface. When adsorption

proceeds from an initially equimolar solution of fibrinogen and serum albumin

(Fig. 34), no albumin is adsorbed. If the initial condition is changed to

a 10:1 concentration ratio favoring serum albumin, as in Fig. 35, then some albumin does adsorb, although it appears to be slowly desorbed. Figure 36

shows that adsorbed serum albumin is quickly displaced by fibrinogen in

solution. Desorption studies (Fig. 37) show that serum albumin does not

desorb at a sufficient rate to account for the rapid displacement by fibrinogen. The conclusion from these studies is that fibrinogen has a much greater

affinity for the surface.




RESULTS AND DISCUSSION

RADIOTRACER RATE STUDIES

Development of Washing Technique

The development of an effective washing technique is of primary

importance in any adsorption investigation. Washing must be effective

in completely removing any clinging solution (carry-out) but should

be as gentle and of as short a duration as possible to minimize desorption.

Protein solutions further complicate the problem by their possible

formation of a denatured film at the air-solution interface which must be

taken into account both in adding and withdrawing sample surfaces. The

experimental approach was to first devise a scheme which would avoid

artifacts from such a denatured film, even at the expense of a time

consuming procedure and a prolonged washing time. Once reproducible results

could be obtained from this system, simplifying changes in the procedure

could be made with these results as a reference point. The reference

technique chosen was to add the protein solution to an adsorption vessel

containing the sample slides covered with a layer of water. After the

desired adsorption time, the entire vessel was rinsed by overflowing with

rinse water.

While this procedure avoids passing slides through the air-solution

Interface, only one adsorption time could be measured per run and the

rinse time was long because of the large volume of protein solution to be

diluted. Several simplifing modifications were found to yield results Identical

to that of the reference technique described above.

1) Slides, contained in 1/8" deep planchets filled with buffer, may be

inserted directly into the protein solution. Hydrophilic surfaces wet

with water may be directly passed through the albumin solution-air

interface. This fact remains to be demonstrated for hydrophobic

surfaces

2) Slides may be removed from the solution by use of the same planchets with the planchets then individually overflowed.

3) Rinsing of slides contained in shallow planchets is generally

inefficient as judged by the time taken for a dye solution to be

cleared. This is due to the difficulty in obtaining mixing in the

thin layer over the top of the slide and from solution trapped between

the slide and the planchet. Rinsing is more efficiently done by

immersing the planchet into a large rinse vessel, removing the slide

from the planchet, and rinsing the slide separately in additional

vessels.

4) The hydrophilic samples may be passed through the albumin solutionair interface without irreversibly carrying out a surface film. Rinsing

in this case must be somewhat more vigorous but conditions can easily

be found that give good agreement with the reference technique. Static

rinsing by dipping into vessels filled with water does not appear to be

sufficient

-64-
1

5) A turbulent flow rinse did not give reproducible results.

While it did seem to minimize washing time, the degree of turbulence

and its distribution over the slide was too difficult to control

precisely.

In summary, the simplest technique suitable for hydrophilic surfaces

and albumin solutions consists of 3 steps:

1) AljDumin solution added to adsorption vessel containing slides

covered with thin film of buffer.

2) Slides removed through air-solution interface.

3) Slides rinsed with flowing water.

The rinse conditions we have chosen are 5 seconds in water flowing

at a rate of one liter per minute at Sl^C. We have not explored the

acceptable limits of these parameters but only moderate adherence to these

conditions has given good reproducibility.

Surface Roughness Determinations

The weight of protein adsorbed per unit area is calculated using

the geometrical area of the counting mask. In order to make meaningful

comparisons of adsorbance on different materials, some measure of the actual

available surface area of the samples must be used. We have measured the

adsorbance of two small molecules, stearic acid and hexadecane, on the various

surfaces and attributed the differences between the surfaces as due to surface

roughness. Similar methods have previously been used as measures of surface

area. For example, Timmons , Patterson, and Lockhart (7l) measured the adsorbance of stearic acid on iron with different polishing treatments. Surface

areas were then calculated by comparing these results with that found on

fire polished glass which was assumed to be perfectly smooth.

Both stearic acid -'"^C and hexadecane --^^C were adsorbed overnight from

benzene solution onto quartz, chromium, and platinum slides. Aliquots of

both solutions were deposited on clean samples of the three materials to

evaluate the relative backscatter efficiency. Relative adsorbance values were used to calculate roughness factors as shown below.

Relative Roughness Factors

Platinum Quartz Chrome

hexadecane 1.00 1.19 1.36

stearic acid 1.00 1.33 1.38

average 1.00 1.26 1.37

-65-

I
The fact that the roughness factors calculated using the non-polar

hexadecane and the polar stearic acid are in good agreement, supports the use of such factors in comparing the various surfaces. However, as such

factors are only relative to the smoothest surface measured and do not

reflect the absolute area available to a protein molecule, the data

presented here have not had such factors applied to them. They may be kept

in mind, but the corrections are relatively small.

Adsorption rate results

Chromium

All results were obtained using the flowing rinse procedure outlined

in the previous section. Figure 38 shows adsorption rates for albumin

on chrome at two different concentrations. Each point is an average of

three samples. The concentration isotherm is flat between concentrations

of 2.5 and 5.0 mg/ml so 3 mg/ml is apparently on the plateau while 0.52

mg/ml is considerably below. The reaction rates vary markedly with concen- tration with the rate of the more concentrated solution almost too fast for our technique to measure. The "equilibrium" adsorbance of 1.7 mg/m^ of the

higher concentration compares favorably with the value of 1.5 mg/m^ found

by ellipsometry as shown in Figure 39. As the ellipsometric adsorbance is

determined in situ, this agreement reinforces the contention that our rinse

procedure is not desorbing appreciable amounts of protein. The apparent

difference in rate is due to the lack of stirring in the ellipsometer cell.

Silica

Figure 40 shows the adsorption isotherm of albumin on fused silica

and Figure 41 gives the adsorption rate for a solution concentration on

the plateau of the isotherm. A semi-logarithmic plot has been used for the

rate data to expand the short time period. The adsorption rate on silica

is slower than that on the chrome; apparent equilibrium adsorbance is not

reached for 10 minutes compared to 10 second for chrome. Additional data

at the plateau adsorbance will be collected in the course of desorption

experiments currently being carried out.

Platinum

Preliminary adsorption measurements on platinum gave equilibrium

adsorbance values of 40 - 50 mg/m^. Such high results prompted

a search for artifacts in connection with this surface. Two possible

sources, backscatter efficiency and surface roughness, have already been

mentioned and the correction factors were much too small to account for

the data. A third possibility, specific adsorption, was tested and appears

to be the responsible phenomenon.

Specific ion adsorption was tested by immersing chromium, quartz,

and platinum slides in a Nal-'--^-'- solution for 18 hours. The slides were

then rinsed, dried, and counted. Both the chrome and quartz showed

-66-

o

LO• C\J

z:

ZD

Z) o

CD

_JX

cr LJ

1^

LO CO

O

LOOO

LO

COO

COO

LO

C\J OO

CsJ O

LOOOO

LO

LU

O

uso

(0

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0)u

cdo

(0

on

<u3

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-68-
CD

^lu / 6uj 'aoNvaaosQv

-69-

very small iodide ion adsorbance while the platinum was over 900 times

higher. To test whether this specific ion adsorption could cause enhanced adsorption of iodine labeled albumin, two labeled albumin

solutions with greatly different specific activities were made up and

used for adsorption measurements on platinum. In the absence of any

enhanced adsorption, the two different activity samples would give the same result. A preferentially adsorbing species would, however, be

present in very different concentrations in the two solutions and the

labeled to unlabeled ratios in the two adsorbed films could well be

different. As the experimental results differed by almost a factor of

two for the two solutions, it is evident that the labeling ratio on the

surface is not the same as in solution and that this difference causes

an overestimation of adsorbance on this surface.

The only solution to this problem is albumin with another label,

perhaps -'^C or H which would not introduce another artifact. Although

platinum would be useful as a reference material, it was not considered

important enough to the program to warrant such an effort and platinum

has been dropped from the radiotracer program. The specific adsorption

noted with this material serves, however, as a warning of the possible

artifacts involved in the use of radiolabeled proteins and the other

surfaces will also be checked for this effect.

-71-
REFERENCES

1. Freund, E. , Med. Jahrb. Wein, 3, 259 (1885).

2. Lyman, D. J., Muir, W. M. , and Lee, I. J., Trans. Amer. Soc. Artif. Int.

Organs, 11, 301 (1965).

3. Bruck, S. D. , Rabin. S., Ferguson, R. S., Biomaterials , Med. Devices and

Artificial Organs J,, 191 (1973).

4. Esnouf, M. P. and Macfarlane, R. G. , in "Advances in Enzymology" (F. F,

Nord, Ed.), 30, p. 255. Interscience , New York (1968).

5. Hulbert, S. F. , King, F. M. , and Klawitter, J. J., J. Biomed. Mater. Res.

Symp., No. 2 (Part 1), 69 (1971).

6. Bruck. S. D. , Biomaterials, Med. Devices and Artificial Organs J., 79 (1973).

7. Bruck, S. D. , Blood Compatible Synthetic Polymers, Charles C. Thomas,

Springfield, Illinois (1974).

8. Brash, J. L. and Lyman, D. J. in "The Chemistry of Biosurfaces" (M. L.

Hair, Ed.) 1, p. 177. Marcel Dekker, Inc., New York (1971).

9. Baier, R. E. , Gott, V. L. , and Feruse, A., Trans. Amer. Soc. Artif. Int.

Organs, 16, 50 (1970). .

10. DePalma, V. A., Baier, R. E. , Ford, J. W. , Gott, V. L. , and Feruse, A.,

J. Biomed. Mater. Res. Symp. No. 3, 6 (4), 37 (1972).

11. Srinivasan, S. and Sawyer, P. N. , J. Colloid Interface Sci., 38^ 484 (19 2)

12. Leininger, R. I., Mirkovitch, V., Beck, R. E. , Andrus, P. G. , and Kolff,

W. J., Trans. Amer. Soc. Artif, Int. Organs, 10, 237 (1964).

13. For example, Vroman, L. and Adams, A. L., Thrombos. Diathes. Haemorrh.

,

18, 510 (1967).

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