NBSIR 75-667

Interaction of Blood Proteins

With Solid Surfaces

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

Polymers Division

Institute for Materials Research

National Bureau of Standards

Washington, D. C. 20234

January 15, 1975

Annual Report for Period

December 1 , 1 973 - October 31 , 1 974

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

NBS-1 14A (REV. 7-73)

U.S. DEPT. OF COMM. BIBLIOGRAPHIC DATA

SHEET

1. PliHl.K'ATlON OR REPORT NO. Reimbursable Agreement - 2

2. Gov't Accession

No.

3. Recipient's Accession No.

i T 1 T I F AND "si ) 1^ T I T I K Interaction of Blood Proteins with Solid Surfaces

5. Publication Date January 15, 1975

6. Performing Organization Code

7. AUTHOR(S) Stromberg, B. W. Morrissey, L. E. Smith,

W. H. arant and C. A. Fenstermaker

8. Performing Organ. Report No. NBSIR 75-667

9. PERFORMING OkGANIZATION NAME AND ADDRESS

NATIONAL BUREAU OF STANDARDS

DEPARTMENT OF COMMERCE

WASHINGTON n C Onj'XA

10. Project/Task/Work Unit No.

11. Contract/Grant No. Interagency Reimbursable;

Agreement

12. Sponsoring Organization Name and Complete Address (Street. City, State, ZIP)

Biomaterials Program

Division of Blood Diseases and Resources

National Heart and Lung Inst., NIH, Bldg. #31, Rm. 5A04

Bethesda, Maryland 20014

13. Type of Report & Period Covered DeC . 1, 1973-

October 31, 1974

14. Sponsoring Agency Code

NHLI

15. SUPPLEMENTARY NOTES

I

I

16. ABSTRACT (A 200-word or less factual summary ot most si^iticant information. If document includes a significant

bibliography or literature survey, mention it here.) The adsorption of blood prOteinS On SUrfaceS haS

been investigated in order to develop a detailed understanding of the initial series of

events that occur when a synthetic material is implanted in the cardiovascular system.

The overall objective of the investigation is to help provide a rational basis for the

characterization and design of materials and the development of test methods. The

relationship between surface charge and protein-surface interaction was investigated

quantitatively by In situ ellipsometry to determine the molecular extension and adsorbance of fibrinogen, serum albumin, and y-globulin on platinum as a function of impressed

surface potential. For all three proteins, no change in adsorbance from the value at

rest potential occurred as the surface potential was progressively made more anodic

until a critical potential was attained, at which time the adsorbance increased significantly. The changes in extension observed as a result of changes in induced surface

potential indicated, however, that conformational changes in the adsorbed layer were

occurring as a result of surface potential. The determination of the bound fraction

(fraction of carbonyl groups directly in contact with the surface) and extension of

adsorbed Y~globulin and g-lactoglobulin as a function of solution concentration indicate

conformational changes with surface population. Similar measurements on yglobulin

crosslinked prior to adsorption indicate that the native conformation exists at low surface coverage. Investigation of the rates of desorption of albumin from silica into

buffer indicates a fast initial desorption followed by a considerably slower desorption

removing most, but not all, of the adsorbed protein during the time periods investigated

17. KEY WORDS (six to twelve entries; alphabetical order; capitalize only the first letter of the first key word unless a proper name; separated by semicolons)

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

protein adsorption; synthetic implants

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[
TABLE OF CONTENTS

Page

ABSTRACT i

SUMMARY 11

INTRODUCTION 1

EXPERIMENTAL 2

Protein Purity 2 Fibrinogen 2 Albumin , 2 Prothrombin 2 y-Globulln 2 B-LdCtoglobulln « 2 Surface Potential 2 Fraction of Free Amino Groups Blocked by DEM 3

Radiotracer 4

RESULTS AND DISCUSSION

MOLECULAR EXTENSION

Surface Potential 6

Carbon 6

Ion Beam Deposited 6

Low Temperature Isotropic 11

BOUND FRACTION

Surface Concentration Induced Conformational Changes 12

Surface Attachments on Hydrophobic Surface 17

RADIOTRACER RATE STUDIES

Silica 19

Polyethylene 19

Platinum. 23

Low Temperature Isotropic Carbon 23

REFERENCES 33

FIGURES

Page

Figure 1: Best fit extension and adsorbance values vs. time for

human fibrinogen at 3.2 mg/ml and pH 7.32 adsorbed on platinum. Applied surface potential indicated between

vertical bars 7

Figure 2: Best fit extension and adsorbance values vs. time for human

serum albumin at 5.0 mg/ml and pH 7.45 adsorbed on platinum.

Applied surface potential indicated between vertical bars. . . 8

Figure 3: Best fit extension and adsorbance values vs. time for bovine

y-globulin at 11.4 mg/ml and pH 7.32 adsorbed on platinum..

Applied surface potential indicated between vertical bars. . . 9

Figure 4: Adsorption isotherm for bovine 3-lactoglobulin at pD 7.4 on silica. Values of bound fraction given for isothermi

points 13

Figure 5: Adsorption isotherm for bovine y-globulin at pD 7.4 on silica.

Values of bound fraction given for isotherm points. . . . . . . 14

Figure 6: Adsorption isotherm for yglobulin cross-linked with diethyl

malonoimidate hydrochloride. Values of bound fraction given

for isotherm points 18

Figure 7: Desorption of serum albumin from silica 20

Figure 8: Desorption and exchange of serum albumin from silica 21

Figure 9: P^te of serum albumin adsorption on polyethylene. Solution

concentration 5.0 mg/ml 22

131 Figure 10: Autoradiograph of HSA- I on LTI carbon 27

131 Figure 11: Autoradiograph of HSA- I on LTI carbon 28

Figure 12: LTI carbon surface 29

Figure 13: LTI carbon surface 29

Figure 14: LTI carbon surface 30

Figure 15: LTI carbon surface 30

Figure 16: LTI carbon surface 31

Figure 17: LTI carbon surface 31

Figure 18: LTI carbon surface 32
TABLES

Page

Table I: Effect of Surface Potential on Protein Adsorption 10

Table II: Extension of Adsorbed y-Globulin 15

131 Table III: Dependence of Adsorbance on Specific Activity of HSA- I. . .24

2 Table IV: Adsorbance of Albumin, mg/m , Estimated using Different

Radioactive Labels 25

I
INTERACTION OF BLOOD PROTEINS WITH SOLID SURFACES

ABSTRACT

The adsorption of blood proteins on surfaces has been Investigated in

order to develop a detailed understanding of the initial series of events

that occur when a synthetic material is implanted in the cardiovascular

system. The overall objective of the investigation is to help provide a rational basis for the characterization and design of materials and the

development of test methods. The relationship betv/een surface potential and

protein-surface interaction was investigated quantitatively by in situ

ellipsometry to determine the molecular extension and adsorhance of fibrinogen, serum albumin, and y-globulin on platinum as a function of impressed surface

potential. For all three proteins, no change in adsorbance from the value

at rest potential occurred as the surface potential was progressively made

more anodic until a critical potential was attained, at which time the

adsorbance increased significantly. The changes in extension observed as

a result of changes in induced surface potential indicated, however, that

conformational changes in the adsorbed layer were occurring as a result

of surface potential. The determination of the bound fraction (fraction of

carbonyl groups directly in contact with the surface) and extension of

adsorbed yglobulin and B-lactoglobulin as a function of solution concentration

indicate conformational changes with surface population. Similar measurements

on y-globulin crosslinked prior to adsorption indicate that the native con- formation exists at low surface coverage. Investigation of the rates of

desorption of albumin from silica into buffer indicates a fast initial

desorption followed by a considerably slower desorption removing most, but

not all, of the adsorbed protein during the time periods investigated.

i
INTERACTION OF BLOOD PROTEINS WITK SOLID SURFACES

SUMMARY _

The adsorption of blood proteins on surfaces has been investigated in

order to develop a detailed understanding of the initial series of events

that occur when a synthetic material is implanted in the cardiovascular

system. The overall objective of the investigation is to help provide a rational basis for the characterization and design of materials and the

development of test methods.

The blood proteins investigated, fibrinogen, prothrombin, yglobulin,

and serum albumin, have been selected either because of implication in the

clotting process or occurrence in high concentration in blood plasma. Special

attention has been given to the purity of these materials and additional

repurif ication has been carried out in our laboratory when necessary. The

major emphasis in this investigation has been the molecular conformational

changes occurring upon interaction of the protein with the surfaces.

The relationship between surface potential and protein-surface interaction

was investigated by quantitative in situ ellipsometry to determine the

molecular extension and adsorbance of fibrinogen, serum albumin, and yglobulin

on platinum as a function of impressed surface potential. For all three proteins,

as the surface potential was progressively made more anodic, no change in

adsorbance from the value at rest potential occurred until a critical potential

was attained, at which the adsorbance increased significantly. The changes in

extension observed as a result of changes in induced surface potential indicated,

however, that conformational changes in the adsorbed layer were occurring as

a result of surface potential.

The previous measurement of the bound fraction (fraction of carbonyl groups

of an adsorbed protein molecule directly in contact with the surface) of prothrombin

and serum albumin on a silica surface showed that approximately ten percent of

these groups were attached to the surface and that this number was independent

of concentration or time. Additional studies have now been made with y-globulin

and 6-lactoglobulin which show a change in bound fraction and extension as a function of solution concentration for these two proteins, inferring a conformational change with surface population. Further investigations with prior crosslinked Y~glot)ulin, to decrease any conformational changes that may occur upon

adsorption, indicate that the native conformation exists at low surface coverage.

Work has been initiated to obtain bound fraction measurement on a hydrophobic

surface. Techniques are being developed and some results are given.

Rates of desorption of albumin from silica have been measured into water

and buffer. In both cases there is a fast initial desorption. In the case of

buffer, this is followed by a second, much slower desorption rate, which removes

most, but not all of the adsorbed protein.

The radiolabeled albumin was used to investigate the apparent porous nature

of a low temperature isotropic carbon surface. High protein concentrations in

small areas of the surface indicated surface scratches or cracks*

11
INTERACTION OF BLOOD PROTEINS WITH SOLID SURFACES

INTRODUCTION

The initial event or series of events that occur when a synthetic

material is implanted in the cardio-vascular system is the adsorption

of components in the blood on the surface of that material. The adsorption

of blood factors and proteins on the surface of the implant can modify

both their biological activity (1,2) and the subsequent interaction of

formed cellular elements ( 3) . It is the intent of this investigation to

develop a detailed understanding of the interaction of four major blood

proteins with surfaces in order to help provide a rational basis for the

design of materials and test methods.

A more extensive rationale for this investigation was given in a previous report ( 4) , together with a detailed description of three major

techniques employed: ellipsometry , infrared difference spectroscopy, and

radiotracer techniques. The work has been expanded during this reporting

period to investigate the possible relationship between surface potential

and thrombosis, the dependence of protein conformation on surface concentration,

and the correlation between ellipsometric and bound fraction measures of

protein conformation. In addition, both ion beam deposited (IBD) and low

temperature isotropic (LTI) carbon were investigated using ellipsometric and

radiotracer techniques.

-1-
EXPERIMENTAL

Protein Purity

Fibrinogen

Bovine and human fibrinogen are repurified in our laboratories by the

Laki ( 5) and Batt ( 6) methods, respectively, to yield a product that is

greater than 96.7% clottable. (The theoretical maximum is 97%.)

Albumin '] ..

The human and bovine serum albumin are obtained as four times recrystallized

material with a purity of' 100%, as determined by electrophoretic analysis.

This material is used as received.

Prothrombin _ -,

; >•

=

•

Cohn Fraction III has been used for most of the work reported here as well

as in our previous report ( 4) . The results on this material have been compared

with an extremely highly purified prothrombin that was kindly supplied to us by Professor Craig Jackson, Washington University. He has determined the

purity by a chromatographic separation and found that there is no detectable

thrombin activity.

y-Globulin ,

. :

• - r

This material is recrystallized and has a purity greater than 95% as determined by electrophoretic analysis. It is used as received.

3-Lactoglobulin

Bovine 6-lactoglobulin, three times recrystallized, was used as received.

Surface Potential

A suitable cell for ellipsometric studies at controlled potentials was constructed by removing the bottom of a fused silica optical cell and replacing

it with a sample holder which allowed a 2 x 5 cm specimen to be clamped to

the base of the cell. A liquid-tight seal v/as made by a butyl rubber gasket.

Electrical contact with the working electrode was accomplished by pressing

a silver strip against the back of the specimen. The counter electrode was

formed by a 16 gauge platinum wire spiral separated from the solution within

the cell by a fine fritted glass disc. A saturated calomel reference electrode,

maintained in a separate compartment, was connected to the cell by an agar-KCl

salt bridge. All adsorption experiments were carried out at 37 °C.

The surface used in all experiments reported here was bright platinum cleaned

in boiling 1:1 concentrated HNO3-H2SO4 and heated at 500°C. The specimen was quenched

in 0.15N NaCl solution, following which the cell was assembled and filled with 0.15N

NaCl as quickly as possible. Human fibrinogen and serum albumin, as well as bovine

-2-
Y-globulin, were obtained from Nutritional Biochemical Co. All solutions were prepared using 0.15N NaCl and the pH was adjusted to 7.4.

The procedure utilized for making the ellipsometric measurements have

been described previously ( 4, 7). In the experiments reported here, the

protein was allowed to adsorb for approximately 120 minutes with no impressed

potential. At this time, the most negative potential of those to be impressed

was applied and ellipsometric readings were taken. The potential was then

incremented in the positive direction at approximately 30 minute intervals, and

and the procedure was repeated until a critical potential, characteristic of

the particular protein, was reached. The data were analyzed using an interative

method ( 8) to determine both the extension and the amount adsorbed.

Fraction of Free Amino Groups Blocked by DEM

The concentration and number of free amino groups blocked by the crosslinking reaction with diethyl malonimidate dihydrochloride (DEM) can be

determined in a straightforward ( 9) manner whenever the extinction coefficient

at 280nm is unchanged as a result of the reaction. Reaction of DEM with serum albumin changed its extinction coefficient. However, since the protein could be

purified by dialysis vs. distilled water and the purified material lyophilized

and subsequently redissolved, gravimetric techniques could be utilized to

determine the extinction coefficient. In the case of y-globulin, however,

dialysis vs. distilled water results in precipitation and loss of the protein.

We have, therefore, utilized ultraviolet optical density, Kjeldahl nitrogen

analysis, and visible optical density follov/ing reaction with ninhydrin for the

cross-linked Y~globulin to determine simultaneously its fraction of nitrogen,

extinction coefficient at 280nm, and the fraction of free amino groups blocked.

These parameters are all interrelated, since the introduction of an unknown

number of cross-links results in a change of both the fraction of nitrogen

and the extinction coefficient.

The resolution of this problem requires the formulation and solution of

three simultaneous equations describing the situation. The extinction coefficient

at 280nm, e , is given by

Certain commercial materials and instruments are identified here and

elsewhere in this publication in order to adequately specify the experimental

materials and procedures. In no case does such identification imply recom- mendation 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.

-3-
where A is the optical density at 280nin of a protein solution subsequently

used for Kjeldahl analysis, L the path length, N the value (in mg N„/inl

protein solution) from Kjeldahl analysis of cross-linked y-globulin, and

^NCL fraction of nitrogen in the cross-linked protein. The value of

f,,_,T is calculated from NCL

M f„ + 14 R F„

= J-N B_

"NCL M + (R/2)F„

p LL B

where >L is the molecular weight of the protein ('^^150, 000 for y-globulin),

the fraction of nitrogen in native material, R the total number of

replaceable amino groups in the protein (120 for y-globulin) , and 'Kq^ the

molecular weight of the cross-link introduced (66 for DEM) . R/2 is the

maximum number of cross-links possible, while F^^ is the actual fraction

of free amino groups blocked by the cross-linking reagent. From the

reaction of ninhydrin ( 9) vrith both native and cross-linked protein,

the fraction of amino groups blocked is '

280/

570 / CL ^^CL\ .570

,280/ / ^CL

F = ^

(3)

B /a280/

' 570 \l '^CL

a280.

where and A^-j^ are the optical densities at 570nm of the native and

cross-linked protein following reaction with ninhydrin, A^ and A^-^ are

the optical densities at 280nm of the native and cross-linked protein

solutions used in ninhydrin reaction, and e^j, is the extinction coefficient

of the native protein at 280nm. For the cross-linked y-globulin prepared

for this study of adsorbed conformation, the simultaneous solution of

Eqs. 1-3 yielded values of e^^ = 1.611, f^^-^ = 0.1672, and F^ = 0.555.

Radiotracer

The radiotracer experiments utilized the same platinum, carbon, and

similarly prepared polyethylene as used for the ellipsometry studies . '^•^I

labeled human albumin was obtained from Squibb, and small samples of serum

albumin and fibrinogen incorporating acetyl groups labeled with tritium were obtained from Dr. Israel Miller of the Weizmann Institute. Isotropic

carbon heart valve discs were supplied to us by General Atomic Corp. Lot

no. 21 C-163 which was used for radiotracer studies had a nominal 10%

-4-
silicon content present as micron sized particles of silicon carbide. The

linear polyethylene, a well characterized polymer, Standard Reference Material

1475, was obtained from the National Bureau of Standards. The quartz and

platinum slides were cleaned with hot 1:2 concentrated MO^:H2S04 for one hour

heat treated at SOO^C for ten minutes, and placed in buffer while

still hot. They were never subsequently exposed to air. The carbon and

polyethylene samples were cleaned by refluxing in a soxlet for several days,

first with ethanol and then water.

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

phosphate buffer and ^^^1 labeled protein was added by syringe.

Adsorption was carried out in a covered glass vessel equipped with a magnetic

stirrer and placed in a thermostatically controlled water bath maintained at

37 + .1 °C. Desorption was carried out on slides on which maximum adsorbance

was attained under the adsorption conditions, as determined by separate

experiments. After adsorption was carried out on a group of slides, they were divided into two sets. One set was rinsed, dried and counted in order

to exactly determine the initial amount of labeled protein adsorbed. The

remaining set was rinsed and transferred, without drying, into a thermostated

vessel containing solvent. After a predetermined desorption time, the slides

were removed, rinsed, dried, and counted. The rate of exchange of adsorbed

labeled protein with unlabeled protein in solution was measured in a similar

manner, except that the slides were transferred to a vessel containing unlabeled

protein of the same solution concentration.

The carbon samples were examined to determine the distribution of labeled

protein remaining after cleaning procedures vjere applied. After adsorption

with HSA--'-31l the samples were scrubbed with detergent solution, rinsed and

dried. Autoradiographic film was applied^ and after suitable periods of

exposure (about 2 months) the films were developed and stripped from the

surface.

131 All counting of I labeled samples was done in a low background beta

counter with a plastic window. Tritium labeled samples were counted without

a window. A mask was used in order to maintain consistent counting geometry,

define the counted area, and eliminate edge effects. In order to establish

the specific activity of the labeled protein solutions, 5 and lOyl aliquots

of the solution were taken and evaporated to dryness on stainless steel

planchets. Separate experiments established that the quartz and chrome

samples had the same backscatter as the steel while the platinum showed 25%

more backscatter. The activities were adjusted for this difference.

-5-
RESULTS AND DISCUSSION

MOLECULAR EXTENSION

Surface Potential

The possible relationship between surface potential and thrombosis

on synthetic implants has been explored by Sawyer and associates (e.g., see

ref. 10). The dependence of protein adsorption on surface potential has been investigated by others using capacitance techniques (11) s cyclic voltainmetry

(12) , potentiostatic and qualitative ellipsometric techniques (13) , and

more recently, internal reflection spectroscopy (14). We have utilized

quantitative in situ ellipsometric techniques to simultaneously determine

both the molecular extension and adsorbance for purified blood proteins

as a function of surface potential.

As shown in Figures 1-3, which are typical of the many experiments performed,

fibrinogen, serum albumin 5 and y-globulin all exhibited a critical potential

at which significant enhanced adsorption on platinum at pH 7,4 compared to the

value at rest potential. In each case^ the adsorbance was unchanged

from the rest potential value as the clamped potential became more anodic, until

the critical potential was reached. At this point, the rate of enhanced adsorption

became approximately proportional to the surface potential. A set of parameters

resulting from the collected data of a number of experiments are shown in Table I.

These results of enhanced adsorbance are in accord with the qualitative

ellipsometric (13) and capacitance (11) measurements, as well as the more

direct internal reflection methods (14)

.

The conformation of all three proteins initially adsorbed at rest potential

was changed by the applied surface potentials o In the case of fibrinogen and

y-globulin, there was a sharp increase in extension when the most cathodic

potencial of the range shown in Table I was applied <> Subsequent experiments with

y-globulin established that the increase in extension was an equilibrium property

and not just a transient one following the initiation of the surface potential

experiment. As the potential became more anodic, the extension in both cases decayed to smaller values. The serum albumin film decreased markedly in thickness

from 10 to 4nm when the critical potential of +0.6V/SCE was applied, resulting in

a more compact film, as judged by a concomitant increase in the index of

refraction.

Carbon

Ion Beam Deposited

Several Ion Beam Deposited (IBD) carbon samples from Whittaker Corp. were

examined to determine if the optical properties of the carbon film were suitable

for an ellipsometric investigation of protein adsorption on this material. IBD

carbon coatings have been termed "diamond-like" because of their durability in

certain applications and because their refractive indices apparently approach

diamond rather than graphite (15) . If the carbon coating were truly "diamond-like"

-6-
ADSORBANCE, mg/m^

LU

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• >

d r-- o d

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QJ T3 4-1

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r-i

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4-1 -H

to CN X)

CU • d

m CO -H

CU

bO

•H

i^u 'NOISN31X3

-7-
ADSORBANCE, mg/m

00 CO CM

1 1 1 1 o

>

CO o

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+

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+

n M

rsi

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ri •H CO e •H

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g

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(+-( fl. CX

0) <: e

4-1

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(U iH

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d a. o c

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(U

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CU

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-8-
ADSORBANCE, mg/m^

CM O 00 CO CM

1 1 1 1 1 1

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A8 0+

\J

0

A90+ - o

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EXTENSION

ADSORBANCE

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0) E t3 u 0) •H 4-t

14-1 « «

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4J iH •H

0) T3

0) 4J C

PQ 03 •H

01

00 •H

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- 0.
•H

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}-i

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4-i o.o

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CO o d P4

4-1 -U X ra M 0) o in

4-) rH ,1?- . ^ d CT3 cC o •H

CU 4-1 O dc^

CO

C 0)

B

•H CC 4-1 vO CN o ba d u PL, s o

•u CO 4-) o 00

PL. <: 0

cyo

CO

"4-1 M

w in 00

CO cc CO o o o

•H \ + + + m 4J > o d o o o

4-1 4-1 4-1

4-1 4J a)

CJ

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(U

14-1 cc

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<4-l Pi 1 W

to

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CO 3

XIOOI >-

-10-
it should have a high index of refraction and very small optical absorption

at visible wavelengths. Both of these properties would be desirable for

ellipsometric applications.

In order to measure an adsorbed protein film, the optical properties

of the substrate must be known accurately. This generally entails the

measurement of the refractive index of the substrate (both real and complex

components) immediately before the surface is covered with protein. In

the case of a composite substrate, this procedure is not sufficient and

additional data must be supplied. If the optical constants (refractive

index) of the uncoated substrate are known, then the thickness and the

real refractive index of the carbon coating could be measured ellipsometrically

The composite substrate would then be completely specified if the carbon film

were non-adsorbing.

IBD carbon films on silicon and polyurethane (Stanford Research Institute

sample 3-425-1-X) were measured ellipsometrically. Samples which were not

beam scanned had a non-uniform film thickness and were not measurable. Beam

scanned samples proved satisfactory. Silicon was chosen as a substrate

because sample to sample variation in the optical constants of silicon have

been found to be relatively small so that the refractive index of uncoated

silicon samples could be used in the calculations necessary for the IBD

coated measurements. Only two variables are measured by ellipsometry so that

only two parameters of a single film can be measured simultaneously. By

measuring the same film on two different substrates, however, another parameter

can be specified. From these measurements it was concluded that IBD carbon

coatings have significant optical adsorbance at 632.8 nm. The measured thicknesses

correspond very well with those estimated by Whittaker Corp. , but would not be

consistent with a high real refractive index and a low complex part. Theoretically,

protein adsorbance measurements could still be made under these conditions, but the

experiments would be involved and additional sources of errors would be introduced.

Low Temperature Isotropic

Prothrombin adsorption on LTI carbon supplied by General Atomic Corp.,

both silicon alloyed and unalloyed, has been measured ellipsometrically. A

highly purified prothrom.bin solution supplied by Prof. Craig Jackson having a con- centration of 0.22 mg/ml, yielded an adsorbance of 2.9 mg/m'^. The thickness

measurements did not vary appreciably with time and were quite reproducible

from sample to sample. The range of extensions observed varied from 4.5 to

8.7 nm with an average value of 6.2 nm. These values are significantly smaller

than those observed on other substrates and the differences are not due to the

high purity protein used for the carbon. For example, an average extension

of 16 nm was measured for the purified prothrombin on chrome compared with

22 nm measured using commercially available protein. The significance of

this low extension must be compared with the observation of very inhomogeneous

albumin adsorption on this carbon reported in the radiotracer section of this

report. The effect of such possible surface porosity on ellipsometric measurements would be expected to be small, but definitive answers must await a further

investigation of the causes of such high local protein concentrations.

-11-
BOUND FRACTION

Surface Concentration Induced Conformational Changes

Speculation based on indirect evidence (16,17), as well as analogies

with protein properties at the air-solution interface, have frequently

led to the assumption that all proteins must "denature" or change drastically

in conformation upon adsorption at the solid-solution interface. In

previous studies utilizing serum albumin and prothrombin (18,4), the molecular

conformation determined from bound fraction measurements was unaffected by

variations of surface concentration along the adsorption isotherm. In

order to clarify this extremely important aspect of protein adsorption,

bound fraction and ellipsometric studies have been carried out on the

adsorbed conformation of g-lactoglobulin and y-globulin. These proteins

were selected on the basis of previous infrared studies of monolayers by Loeb (19)

and the potentiometric titration results of Kochwa and coworkers (20) » both of

which indicated that changes in conformation paralleled changes in the surface

concentration. . " r^.i

The adsorption isotherms of native 6-lactoglobulin and y-globulin

adsorbed on silica at pD 7.4 are shown in Figures 4 and 5. The values given

along the curves represent the experimentally measured bound fraction (the

fraction of carbonyl groups of an adsorbed protein molecule directly interacting with the surface) at that equilibrium concentration. In each case,

it is evident that there is a significant decrease in the bound fraction

with increasing adsorbance. For 3-lactoglobulin , the decrease in bound

fraction from 0.14 to 0.01 corresponds to a decrease from 30 to 2 carbonyl

surface attachments per molecule, while for y-globulin, the change of bound fraction

from 0.2 to 0.02 results in a decrease from 270 to 30 contacts.

To further investigate these changes in conformation of y-globulin and

3-lactoglobulin, we have determined the extension of the adsorbed film on planar fused silica using ellipsometry. The protein solution concentrations

were selected to correspond to those regions of the adsorption isotherms

(Figures 4 and 5), which exhibited the largest differences in the bound

fraction. As shown in Table II, the extension of adsorbed y-globulin

increased with increasing concentration of the solution. At concentrations

of 0.11 mg/ml and 10.0 mg/ml, bound fractions of 0.16 and 0.02, respectively,

were measured for the adsorbed y-globulin, while extensions of 19 and 64 nm were observed, respectively. Similar ellipsometric studies utilizing 3-

lactoglobulin were unsuccessful. We can only spectulate that the relatively

small size of the 3-lactoglobulin molecule does not result in a sufficient

difference in index of refraction to define the adsorbed layer.

The decreasing bound fraction (number of surface attachments) of y-globulin

with increasing adsorbance has been reported also for a number of synthetic,

random coil polymers. Using infrared bound fraction measurements to study the

adsorbed conformation of poly(methyl methacrylate) and polystyrene on silica,

Thies (21) observed a decreasing number of surface attachments with increasing

-12-
,1 1 0

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-14-
Table II

Extension of Adsorbed y-Globulin

Native

Cone. Extension Bound

mg/ml nm Fraction

0.11 19 0.16

0.20 32 ^0.12

0.28 27 'vO.12

10.0 64 0.02

DEM Crosslinked

0.30 21 0.06

10.7 20 >0.01

-15-
adsorbance. Similarly, Fontana (22) showed that the bound fraction of an alkyl methacrylate-polyglycol methacrylate copolymer decreased with increasing

adsorbance. The ellipsometrically measured extension of an adsorbed molecule

normal to a surface has been found to increase with time during the adsorption

period for the adsorption of polystyrene (23,24) and polyethylene glycol (25).

The increase in extension, as well as the decrease in the bound fraction, with

increasing surface concentration have been interpreted to result from a decrease

in the number of surface attachments. The similarity of these results to

those reported for yglohulin and 3-lactoglobulin suggests that both proteins

undergo conf oriuational changes. The agreement between changes in bound fraction

and extension for adsorbed yglobulin supports the proposed conformational

change.

The similarity of the results for synthetic polymers and Y~globulln

implies that the protein is rather pliant. Biologically, this feature

is undoubtingly important, since in solution;, 7S yglohulin is an important

antibody which must interact with a wide variety of antigens. Furthermore,

recent studies by Kim and coworkers (26) have shown that prior adsorption of

y-globulin results in the significant adhesion of platelets to a surface.

Such effects may indeed depend on the adsorbed conformation.

The bound fraction and ellipsometric conformation studies of B-lactoglobulin and y-globulin are entirely consistent with previous findings

(19,20). Loeb demonstrated a change in the ratio of the intensity of the

infrared bands characteristic of a-helical and 3-sheet structures for

3-lactoglobulin in a transferred film as a function of surface pressure

in the spread monolayer. This finding was interpreted as a "denaturation"

at low surface concentration, although the introduction of artifacts due

to drying and working vjith collapsed films make it difficult to establish

whether the molecule was folding or unfolding as it "denatured". Kochwa's

in situ potentiometric titration study of the adsorption of y-globulin to

a polystyrene latex showed that additional titrable groups per adsorbed

molecule appeared at low surface concentration. The strong correlation

between surface concentration and acid uptake established a definite change

in conformation upon adsorption as a function of the adsorbance.

None of these results (potentiometric titrations, infrared spectroscopy

of transferred monolayers, ellipsometric, or bound fraction) for native

Y-globulin and 3-lactoglobulin actually establish at what point along the

adsorption isotherm the adsorbed protein is "native". While analogies to

air-liquid adsorption studies where the low concentration, low pressure

state coincides with a spread monolayer have frequently been made, studies

of polyelectrolytes adsorbed at the solid-liquid interface (27) establish

that adsorbate-adsorbate interactions generally determine both the adsorbance

and conformation. It is therefore possible that major distortions of the

native protein conformation could occur at high, rather than at low, adsorbance.

To further investigate these changes in y-globulin conformation, as well as the question of which adsorbed conformation corresponds to that

in solution, bound fraction and ellipsometric studies were carried out using

the cross-linked protein. Diethyl malonoimidate hydrochloride (DEM) was reacted with y-globulin as previously described (18) to block 56% of the

-16-
free amino groups (see Experimental section, p. 3).

The adsorption isotherm of cross-linked DEM-y-globulin is shown in

Figure 6. The values given along the curve represent the experimentally

measured bound fraction at that equilibrium concentration. Compared to

native y-globulin (Fig. 5), there are approximately one half the number of

carbonyl attachments at a given equilibrium concentration. The extension

of the adsorbed cross-linked protein is given in Table II at two concentrations approximately equal to those used to study native protein. At 0.30 mg/ml

and 10.7 mg/ml, the extensions are experimentally indistinguishable from each other

and closely resemble the results for low concentration native protein. Thus, while

the bound fraction results indicate that cross-linked yglobulin is still somewhat

flexible, with an adsorbed conformation intermediate between those at high and low

surface concentrations, the ellipsometric results indicate that the conformation

at low surface concentration is that of the native protein. It would appear that

if the cross-linking reaction is "freezing" the native, solution conformation, then

the native conformation exists at low surface coverage and the "denaturing" force

is the protein-protein interactions between adsorbed molecules.

Caution must always be exercised when comparing bound fraction and

ellipsometric results for different proteins. It is difficult to establish

correlations between the techniques and draw conclusions concerning conformational

changes of adsorbed proteins. Native and cross-linked y-globulin yield similar

extensions at low concentration, despite the significant difference in bound

fraction. It is quite conceivable that this could occur, since the introduction

of additional constraints on internal motion would certainly limit local re- arrangements in the vicinity of the surface, without changing the overall molecular

dimensions. Only for a flexible, random-coil polymer would one expect in general

a correlation between the number of surface attachments and the extension. Even

in this case, however, unusual adsorbed conformations could destroy the correlation.

Surface Attachments on Hydrophobic Surface

2 A monodisperse polystyrene latex of 91 nm particles (63 m /g) was purified using mixed ion-exchange resins (28) and techniques developed for

its use as an adsorbent for infrared bound fraction measurements. Since

all adsorption suspensions must be prepared by pipetting protein solution

and latex, the errors inherent in the procedure are somewhat greater than

in the case of silica.

The results of experiments to date define an isotherm for bovine serum

albumin with an apparent plateau adsorbance of lAOmg protein per gram of

polystyrene latex at an equilibrium concentration of "^2.0 mg/ml. This

adsorbance is some 2.5 times greater per unit area than that occurring on

silica. The values of the bound fraction, while generally higher than on silica, show a large scatter. Efforts are in progress to refine the technique

and reduce the uncertainties.

-17-
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-18-
RADIOTRACER RATE STUDIES

The success of an investigation of the rates of adsorption, desorption

and exchange of blood proteins is dependent, in part, on the development

of techniques that will be effective in completely removing any "carry-out"

from the adsorption solution and will prevent coating the slide with denatured

protein that may exist at the solution-air interface. This problem and

the techniques employed here were discussed in some detail in our last annual

report ( 4) . An unexpected problem, preferential or specific adsorption of the

labeling atom, was also observed for platinum, one of the materials investigated.

This problem has been investigated somewhat further and is discussed below.

Finally, rates of adsorption for human serum albumin on chromium and silica,

shown in our last report are not given again here, but are used as the basis

for our desorption and exchange results.

Silica

The rate of desorption of albumin into water from fused silica is shown

in Figure 7. A very rapid initial rate of desorption occurs; about 30

to 35% of the adsorbed material is removed within approximately one minute.

Subsequently, there is no further change in the amount of protein remaining

for time periods up to two hours. The desorption of albumin into

buffer, as seen in Figure 8, however, indicates a somewhat different behavior.

A similar rapid initial desorption is also observed, although it appears to

continue for a longer time period with more protein removed. A second, slower, desorption rate also occurs over longer time periods. In this case approxi- mately 50% was desorbed within the first five minutes, with an additional 30%

removed in the subsequent four hour period. The second rate may be a function

of the ionic strength of the solvent.

h Also investigated was the rate of exchange of adsorbed (labeled) albumin

with albumin (nonlabeled) in solution. Under the conditions of the experiment,

the total quantity of albumin on the surface should be constant. Shown in

Figure 8 is the amount of initially adsorbed albumin, that remained on the

surface. This is seen to be, within experimental error, the same as the amount

remaining after desorption into buffer for the system and time period investigated,

Xne sxiuiJ-aricy oecween rates of desorption and exchange were also observed for

a random coil synthetic macromolecule , polystyrene (29).

Polyethylene

131

Measurements of HSA- I adsorbance on polyethylene as shown in Figure 9,

show that the apparent equilibrium adsorbance is not reached for 15 min.

compared to 10 sec. for chrome and 15 minutes for silica reported previously

( 4). The differences in rates to plateau values may be related to the surface

free energy, i.e., as the surface energy decreases, the times to plateau decrease..

An increase in the extension with decreasing surface energy was noted for

several blood proteins (4).

-19-


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-22-
Platinum

In our last report ( 4) we discussed the abnormally high apparent

adsorbance values obtained on platinum with HSA-"'--^-'-! and described the

131 results of the use of solutions of different activities of I to test

for its adsorption. Additional work has verified that platinum interacts

with I to give erronous results when this atom is used to label the

protein. Other materials were also investigated to determine whether

they were also subject to specific adsorption of -^-^-^I. The results are shown in Table III for three polymeric materials. Unlike the platinum,

the adsorbance values are independent of the specific activity for these

three polymers. Similar results were obtained for silica and chrome.

Comparison was made of adsorption on platinum (and chromium as a reference) between HSA--'--^-'-I and albumin labeled with tritium-containing

acetyl groups. As can be seen in Table IV, the chromium measurements

agreed well, but the adsorbance measured by the tritiated albumin on platinum was less than 5% of the apparent adsorbance value obtained using

the iodinated albumin. This is a fairly direct conformation of our hypothesis of enhanced specific adsorption induced by the incorporation

of iodine into the albumin.

Low Temperature Isotropic Carbon

131 The adsorption of HSA I on low temperature isotropic carbon (LTI)

has been studied. All results were obtained using the same experimental

procedure as outlined in the previous section. An initial study showed

an equilibrium adsorbance of 12.9 mg/m on the carbon surface, but only

about 3 mg/m was removed by extensive scrubbing with detergent solutions.

This same scrubbing treatment has generally been successful in removing

all of the adsorbed albumin from other surfaces, such as quartz and

platinum. This suggests that porosity of the surface might be suspected

as being responsible for both the rather high adsorbance values and the

difficulty in desorption. Porosity of the surface might arise from a generalized homogeneous distribution of vacancies on the scale of atomic

or crystallite dimensions or, alternatively, porosity might be centered

in defect structures of a larger scale and would be distributed inhomogeneously across the surface. Existing information about the LTI carbon

indicates that the first possibility is somewhat remote, thus attempts

were made to experimentally evaluate the possibility of inhomogeneous

adsorption associated with larger scale porosity.

Figure 10 shows a typical radiograph where the dark spots indicate

a high concentration of serum albumin. The small white spots are pin holes

in the film and the large white area in the lower right is a portion of the

film that was not in contact with the surface and, therefore, serves as a blank. Figure 11 shovrs a larger portion of an exposed film for a second

carbon sample of the same lot under similar experimental conditions. The

general gray background indicates a rather homogeneously distributed component

of the adsorbed protein that is present on all areas of the carbon surface

and was not removed by the detergent washing. That this gray background is

due to radiolabeled protein and not light exposure or development fog is seen by comparison with the white area in Figure 10. The very dark spots

-23-
Table III. Dependence of Adsorbance on HSA-"^I

Specific Activity of

Polyethylene

Relative Polyethylene Silicone Terephthalate

Activity mg/m^ mg/m^ mg/m^

1.00 5.6 6.1 6.0

0.67 .4.4 7.2 5.4

0.50 . .„ 5.4 6.5 5.7

0.33 4.7 ' 6.5 ^ . ; 6.1

0.25 . 5.3 5.2 5.6

0.20 4.6 ,. ' - : - 5.7

-24-
Table IV. Adsorbance of Albumin, mg/m , Estimated using

Different Radioactive Labels

Pt. Cr.

mg/m^ mg/m'

3

H -acetyl group 3.56 3.75

label

'^''"I label 77.0 2.67

-25-
scattered across the film indicate that a large fraction of the measured

adsorbance is associated with very high protein concentrations in small areas. Enlargements of several selected areas of the film that include some of these

very dark areas are shown in Figures 12-15. The structure in these areas is

striking in that many seem associated with surface scratches or cracks. This

is especially apparent in Figures 14 and 15 by the "beads on a string" appearance

of the autoradiograph, suggesting that sub-surface porosity may have been opened

up by a scratch or crack.

The carbon surface underlying one of the radiographs is shown in Figure 16

at the same magnification as the film seen in Figure 10. At this level the

surface seems more or less homogeneous with small hills and depressions and

a few concentric rings. Alignment of the film with the surface from which

it was stripped showed no correspondence of the dark spots due to high proteins

concentration with these concentric rings. Higher magnification of the carbon

surface are shown in Figures 17 and 18. The magnification in Figure 17 corresponds

rather closely to that of the autoradiographs in Figures 12-15. While the carbon

surface is covered with numerous scratches and cracks at this scale, they are much smaller than the features which are disclosed by the protein adsorbance.

Further autoradiographs will be taken that will include reference marks to

permit exact alignment of the film with the surface. This should permit a better identification of the surface features responsible for the high protein

concentration.

-26-
0

131 Figure 10: Autoradiograph of HSA- I on LTI carbon

-27-
131

Figure 11: Autoradiograph of HSA- I on LTI carbon

-28-
Figure 12: LTI carbon surface

Figure 13: LTI carbon surface

-29-
lOOum

Figure 14: LTI carbon surface

lOOum,

Figure 15: LTI carbon surface

-30-
0.2cm

-31-
100 ym

Figure 18: LTI carbon surface

-32-
REFERENCES

1. Gitel, S. N., Owen, W. G. , Esman, C. T. , and Jackson, C. M. , Proc

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(1974).

19. Loeb, G. I., J. Polymer Sci., 34C, 63 (1971).

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-33-
21. Thies, C, J. Phys. Chem. , 7£, 3783 (1966).

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and Olsen, D. , Trans. ASAIO, 20, 449 (1974).

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USCOMM-IMBS-DC' -34-

j