NBSIR 76-1128

Interaction of Blood Proteins

with Solid Surfaces

L. E. Smith, R. E. Dehl, W. H. Grant, R. R. Stromberg and B. W. Morrissey

Polymers Division

Institute for Materials Research

National Bureau of Standards

Washington, D. C. 20234

August 1 6, 1 976

Annual Report for Period

November 1, 1975 - August 15, 1976

Issued October 1976

Prepared for Biomaterials Program

Division of Heart and Vascular Diseases

National Heart and Lung Institute

National Institutes of Health

Bethesda, Maryland 20014

Interagency Reimbursable Agreement
NOTICE

This is a draft of a report prepared as an account of progress of work

sponsored by the National Heart and Lung Institute. It is not to be

construed as a report, opinion, or recommendation of the National Heart

and Lung Institute. When produced in final form it will be available

from the National Technical Information Service, 5285 Port Royal Road,

Springfield, Virginia 22151.

Certain commercial equipment is identified in this report 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 identified

is the best available.
NBSIR 76-1128

INTERACTION OF BLOOD PROTEINS

WITH SOLID SURFACES

L E Smith, R E Dehl, W. H Grant, R R Stromberg and B. W. Morrissey

Polymers Division

Institute for Materials Research

National Bureau of Standards

Washington, D C 20234

August 1 6, 1 976

Annual Report for Period

November 1, 1975 - August 15, 1976

Issued October 1 976

Prepared for Biomaterials Program

Division of Heart and Vascular Diseases

National Heart and Lung Institute

National Institutes of Health

Bethesda, Maryland 20014

Interagency Reimbursable Agreement

U S. DEPARTMENT OF COMMERCE, Elliot L. Richardson, Secretary

Edward O. Vetter, Under Secretary

Dr Betsy Ancker-Johnson, Assistant Secretary for Science and Technology

NATIONAL BUREAU OF STANDARDS, Ernest Ambler, Acting Director

BIBLIOGRAPHIC data 1. Report No. 2. SHEET 4

3. -Recipient':; Accession No.

4. Title and Subtitle INTERACTION OF BLOOD PROTEINS WITH SOLID SURFACES

5. Report Date August 16, 1976

6.

7. Author(s) L. E. Smith, R. E. Dehl, W. H. Grant, R. R. Stromberg

and B. W. Morrissey

8. Performing Organization Kept.

No * NBSIR 76-1128

9, Performing Organization Name and Address

; Polymers Division

Institute for Materials Research

National Bureau of Standards

Washington, D. C. 20234

10. Pro jeet/' I sk/Work Unit No.

11. Contract T-m’ 'R

Y01-HB- 3-0014

12. Sponsoring Organization Name and Address

Devices and Technology Branch, Biomaterials Program

Division of Heart and Vascular Diseases

NATIONAL HEART AND LUNG INSTITUTE

Bethesda, Maryland 20014

13. Type of Report & Period

Covered Nqv> 1} 1975 _ August 15, 1976

14.

15. Supplementary Notes

I 16. Abstracts Inis investigation is uesigned to help characterise the sur:^. y -opertie'. o: tardiofascuxar implant materials. Such properties largely determine t.:> success cr failure e! implants and may therefore be used as the basis for in vitro test methods. TLt focus ol

tais investigation has been on the measurement of protein adsorption on surfaces,

emphasizing measurements of the conformational changes which occur upon adsorption.

During this reporting period, several materials produced by contractors of the NHLi EioKiattrials Program have been examined by ellipsometry and a number have been found .citable for further work. Conformational changes at very short adsorption times are of

considerable importance and preliminary results indicate that our techniques are capable

ut measurements at times as short as two seconds. We have analyzed commercially available radiolabeled human serum albumin and found considerable amounts of dimers and higher

aggregates in some commercial preparations. We have begun to assess the errors involved

in £he use of unfractionated proteins for adsorDtiun measurements by using pur.. free

17. Key Words and Document Analysis. 17a. Descriptors

) Adsorption

Blood protein

Ellipsometry

Implants

Polymer adsorption

Protein adsorption

synthetic implants

17b. Identif iers/Open-Ended Terms

17c. COS ATI Field Group

is. Availability .Statement Releasable to the public. 19. ei. ur it y ( la • i 1 1 1 1 . 21. No. oi i s Order from: National Technical Information Service

c por c )

I N( I. V' .11 1! I'* 42 1 5285 Port Royal Road 20. Security C . i ,i •

. , ( I ins 22. I’ncc Sprinofield, Vircrinia 22151 1 Nf i c |

!

'

!

1 D $4.00

F°f<M ntis-33 rev i -7.. | \| h iRS 1.

1

) HY V. 1 AND 1 . \ KS( .! > THIS FORM \1.\V Hi-. RiTROl.l ( i.i) LI 4 C OMM- . ' ; *.

monomer and aggregates. The use of ligit

mattering to measure the conformation of

piotein adsorbed on small particles has been

evaluated in a study of y-globulin adsorption

on polystyrene. The results agree well no th

similar measurements made by ellipsometr .

TABLE OF CONTENTS

' Page

ABSTRACT i

SUMMARY ii

INTRODUCTION 1

EXPERIMENTAL 1

Fibrinogen Purity 1 Ellipsometry 2 NHLI Contractor Biomaterials 2 Radiotracers 2 Protein Purity 3

RESULTS AND DISCUSSION 3

EXAMINATION OF NHLI CONTRACTOR BIOMATERIALS 3

Polyether urethanes (Stanford Research Institute) 3 Ethylcellulose perf luorobutyrate (Midwest Research Institute). . . 6

Polyacrylamide hydrogel (Franklin Institute Research Laboratories) 6

Polyacrylamide hydrogel (Polysciences, Inc.) 6

CURRENT STATUS OF OTHER NHLI CONTRACTOR BIOMATERIALS 7

Glow discharge coatings (Research Triangle Institute) 7 Polyalkylsulfone (A. D. Little) 7 Ion-plated carbon on stainless steel (General Atomic Corp.). ... 7 Heparin bonded to silastic-coated glass (Carnegie-Mellon Univ.). . 7

RADIOTRACER ADSORPTION STUDIES 8

Effect of Molecular Impurities on Adsorption of HSA 8

Adsorption Isotherms of HSA 33 -*-I Monomer and Dimer on Polyethylene 13

FIBRINOGEN ADSORPTION STUDIED BY AUTOMATIC ELLIPSOMETRY 29

ADSORBED PROTEIN CONFORMATION BY LIGHT SCATTERING 30

REFERENCES 34

APPENDIX 35

FIGURES

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Page

/ 1: Extension of human fibrinogen on SRI Polyether

Urethane 3-2000-1-E 4

2: Extension of human fibrinogen on SRI Polyether

Urethane 3-1000/425 ( 70/ 30)-l-X) 4

3: Adsorbance of human fibrinogen on SRI Polyether

Urethane 3-2000-1-E 5

4: Adsorbance of human fibrinogen on SRI Polyether

Urethane 3-1000/425(70/30)-l-X 5 131

5: Gel Permeation Chromatogram of I HSA 9 125

6: Gel Permeation Chromatogram of I HSA 10

125

7: Relative Radioactivity of the I HSA Chromatogram

Shown in Figure 2 11

125

8: Gel Permeation Chromatogram of I HSA 12

9: Gel Permeation Chromatogram of Unlabeled HSA 14

125

10: Adsorption of I HSA Fractions on Polyethylene .... 16

125

11: Adsorption of I HSA Monomer on Polyethylene in Presence

of Aggregates 17

131

12: Adsorption of the Monomer of I HSA on Polyethylene . . 18

131

13: Adsorption of the Monomer of I HSA on Polyethylene . . 19

131

14: Adsorption of the Monomer of I HSA on Polyethylene . . 20

131

15: Adsorption of the Monomer of I HSA on Polyethylene . . 21

131

16: Adsorption of the Monomer of I HSA on Polyethylene . . 22

131

17: Adsorption Isotherm for the Monomer of I on Polyethylene 23

131

18: Adsorption of the Dimer of I HSA on Polyethylene ... 24

131

19: Adsorption of the Dimer of I HSA on Polyethylene ... 25

131

20: Adsorption of the Dimer of I HSA on Polyethylene ... 26

FIGURES

Page

Figure 21: Adsorption of the Dimer of I HSA on Polyethylene . . . 27

131 Figure 22: Adsorption Isotherm for the Dimer of I HSA

on Polyethylene 28

Figure 23: Width at half height (HWD) of Scattered Frequency

Spectrum vs. q

2 /Pi (q = scattering wave vector) for

Surfactant Free Polystyrene Latex 31

Figure 24: Radius of Polystyrene Particle with Adsorbed Human

y-Globulin vs. Time 33

TABLES

Table I: Adsorbance of Selected Fractions of Labeled and Unlabeled

HSA on Polyethylene and Silica 15

ABSTRACT

This investigation is designed to help characterize the surface properties

of cardiovascular implant materials. Such properties largely determine the success or failure of implants and may therefore be used as the basis for

in vitro test methods. The focus of this investigation has been on the

measurement of protein adsorption on surfaces, emphasizing measurements of

the conformational changes which occur upon adsorption. During this

reporting period, several materials produced by contractors of the NHLI

Biomaterials Program have been examined by ellipsometry and a number have

been found suitable for further work. Conformational changes at very short

adsorption times are of considerable importance and preliminary results

indicate that our techniques are capable of measurements at times as short

as two seconds. We have analyzed commercially available radiolabeled human

serum albumin and found considerable amounts of dimers and higher aggregates

in some commercial preparations. We have begun to assess the errors involved

in the use of unfractionated proteins for adsorption measurements by using

purified monomer and aggregates. The use of light scattering to measure the

conformation of protein adsorbed on small particles has been evaluated in a study of y-globulin adsorption on polystyrene. The results agree well with

similar measurements made by ellipsometry.

1
SUMMARY

This investigation is designed to aid in the characterization of the

surface properties of materials used as implants in the cardiovascular

system. Surface properties play a major role in determining the success of

an implant and therefore should form the basis of in vitro test methods to

select and develop useful materials.

The major emphasis of the work has been on the measurement of conformational change that occurs in the protein upon adsorption. Ellipsometry

has proved successful in this regard when applied to several model surfaces.

During the current reporting period a number of materials produced by

contractors of the NHLI Biomaterials Program and considered of potential

clinical importance by the Project Officer have been surveyed. Several

materials were found to be suitable for fibrinogen adsorption measurements.

In addition, carbon coated samples (ion-beam deposited, ion plated, and vacuum deposited) are suitable if the carbon coating is thicker than 350 nm,

which would be the case for coatings of current biomaterial application.

Measurements of adsorbed protein conformation on these materials can be

compared with in vivo test results and any correlation will guide future

work in the development of useful test methods.

The conformation of the protein as it is first adsorbed and the changes

in conformation that it undergoes as the surface population increases are important parts in the sequence of events occurring when blood contacts a foreign surface. We have developed techniques for the measurement of such

conformational changes at short adsorption times (~2s). Thus far we have

been unable to detect conformational changes on this time scale in fibrinogen

adsorbed on platinum or silica substrates.

The use of radiolabeled protein for adsorption measurements without

careful purification of both the labeled and unlabeled components can lead

to confusing and potentially misleading data due to the complex competitive

adsorption between the protein monomers, aggregates and polypeptide fragments

that are generally present. We have concentrated on this point during this

reporting period and have analyzed some commercially available radiolabeled

human serum albumin (HSA) samples. There are differences in composition from

different manufacturers. Self-radiolysis does not seem to be an important

contributor to the production of polypeptide fragments that may be present.

We have used purified fractions of HSA-monomer and of HSA-dimer to measure

the adsorption and competitive adsorption of the monomer and dimer on a polyethylene surface.

The use of quasielastic light scattering to measure the conformation of

protein adsorbed on small particles has been investigated. The method is

capable of high precision and the results of y-globulin adsorption on polystyrene compares favorably with similar measurements made by ellipsometry.

ii
INTERACTION OF BLOOD PROTEINS WITH SOLID SURFACES

INTRODUCTION

The adsorption of plasma proteins is the first in a complex series of

events that occurs when a synthetic material is placed in the cardiovascular

system. It is known that this protein adsorption can alter the course of

subsequent reactions at the implant surface but the mechanism by which this occurs is not well understood. If the detailed behavior of proteins adsorbed

at the solid surface were known and capable of control, the fate of artificial

materials placed in body could be predicted with greater certainty and more successful materials perhaps could be designed. This investigation is

directed toward the identification of factors capable of affecting protein

adsorption and the development of a description of the changes that occur

in the protein upon interaction with a surface.

A major portion of the research effort this year has been directed

toward the examination of potentially useful synthetic biomaterials provided

by contractors to the NHLI Biomaterials Program. Where possible, the

adsorption of fibrinogen from solution onto these materials has been studied

by ellipsometry . It is anticipated that as this data is accumulated,

significant differences either in the rate of adsorption, the total quantity

adsorbed, or the molecular conformation of fibrinogen on these various

surfaces will lead to useful correlations with their blood compatibility

as implant materials. This might provide the basis for a rapid in vitro

screening test.

Experimental artifacts due to the use of impure radiolabeled proteins

have led many workers to erroneous conclusions in the past about the

adsorption of these proteins. We have conducted radiotracer studies of

labeled HSA on polyethylene and silica surfaces, using carefully fractionated

HSA. The adsorption of fractionated HSA monomer and dimer have been separately

examined.

EXPERIMENTAL

Fibrinogen Purity

Human fibrinogen was used in these experiments. It was purified by

the Batt (1) modification of the Laki (2) method to yield a product which

is greater than 96.7% clottable. (The theoretical maximum is 97%). At
the suggestion of Prof. David Waugh of MIT, we are now preparing purified

fibrinogen in fairly large quantities, fast-freezing the solution batches

in liquid nitrogen, and storing the frozen solutions in dry ice until they

are needed. With these precautions, the fibrinogen purity can be assured

for relatively long periods of time.

Ellipsometry

The techniques of ellipsometry, methods of sample handling, determination

of the optical constants of the substrate, etc., have been described in

previous Annual Reports (3,4). In all cases, the fibrinogen adsorption

experiments were performed at 37°C. Because many of the NHLI Contractor

Biomaterial samples were elastomeric, it was necessary to construct a very

heavy Pt weight to hold the sample flat and prevent movement of the sample

due to relaxation during the experiment, and still permit light to reach

the sample surface.

For the preliminary adsorption experiments with the automatic ellipsometer

described in this report, a straightforward method of introducing the protein

solutions was adopted. After determining the optical constants of the

substrate under buffer solution, the solution was withdrawn from the ellipsometer

cell as completely as possible with a syringe, leaving a thin film of solution

on top of the (hydrophilic) substrate. A premeasured quantity of filtered

protein solution was then quickly poured into the cell. Since the solution

is introduced in a highly turbulent manner, it should mix rapidly with any

residual buffer solution in the cell. This technique allowed measurement of

the ellipsometric parameters in less than 2 seconds after the solution pouring

was begun. The resultant changes in the parameters were smooth and monotonic

through the total observation times, which ranged up to 3 hours.

NHLI Contractor Biomaterials

The NHLI Contractor Biomaterials which we have received this year were shipped to us in sterile water, in the case of the hydrogels, or in sterilized

packages, in the case of uncoated substrates. These precautions should

eliminate problems that we have had with some samples in the past, due to

bacterial growth in nonsterile aqueous media. For some samples which may be

porous as well as hydrophilic, we allow the substrate to soak in buffer

solution overnight before the final measurement of its refractive index.

This procedure assures that the index will not be changing during the time

that protein is being adsorbed, since such changes could lead to serious errors

in interpretation of the data.

Radiotracers

A different experimental arrangement for the adsorption of the labeled

proteins on surfaces was designed to decrease the volume of solution necessary

for an adsorption experiment. Prior to this report an adsorption solution

2
consisted of non-labeled proteins diluted with labeled proteins and thus

large volumes were easily obtainable. In the present case, the solution

contains only labeled proteins and has a smaller total volume to compensate

for the higher cost of using pure labeled proteins. This modified

experimental arrangement is confined to the adsorption vessel. The vessel,

which has a total volume of 10 ml is equipped with a plunger that is designed

to hold two slides under the solution and also to aid in their removal from

the solution. The adsorption vessel is shaken with a mechanical shaker in

a thermostatically controlled water bath maintained at 37 ± ,1°C. All

other experimental procedures are the same as described in previous Annual

Reports

.

Protein Purity

The commercially labeled proteins obtained from Squibb Pharmaceutical

Co. and Amersham/Searle and the non-labeled proteins obtained from

Nutritional Biochemicals Corp. were further purified by passing these

materials through a Sephadex G-200 gel permeation chromatographic column

which separated out the trimer, dimer, monomer and low molecular weight

polypeptide.

RESULTS AND DISCUSSION

EXAMINATION OF NHLI CONTRACTOR BIOMATERIALS

Polyether Urethanes (Stanford Research Institute)

We have completed the study of fibrinogen adsorption onto SRI polyether

urethanes (A) 3-2000-1-E and (B) 3-1000/425(70/30)-l-X. The results of 3

separate adsorption experiments with each material are shown in Figures

1-4. No significant changes were observed in either the protein film

thickness (extension) or adsorbance during the time interval from about

0.5 to 3 or 4 hours. The (B) substrate is mechanically stiffer than the (A)

substrate, and unlike the latter it did not absorb water and become

translucent after immersion in water for a day or more. Despite these

differences, however, the (A) and (B) substrates appear to have a very

similar affinity for fibrinogen, adsorbed from solution. The average

fibrinogen film thickness calculated from the data of Figure 1 for the

(A) material is 30.7 ±4.6 nm, while the (B) substrate data of Figure 2 yields an average thickness of 32.0 ± 5.7 nm. The average adsorbance on

the (A) and (B) substrates, calculated from the data shown in Figures 3

and 4 are, respectively, 4.75 ± 0.23 mg/m2 and 5.04 ± 0.39 mg/m2. Thus,

within experimental error, there is no difference in the equilibrium film

thickness or adsorbance of fibrinogen on the two SRI substrates. However,

the rate of protein adsorption during the initial half hour remains as a possible difference between the two substrates.

3
EXTENSION,

nm

50 T T

40 -

30 -

20 -

10 -

0

m

+ +

A

0

+

A

0

SRI Polyether Urethane

3-2000-1 -E

^Fibrinogen, 3.0 mg/ml

0 50 100 150 .200 250 300

TIME, min

Figure 1: Extension of human fibrinogen on SRI Polyether

Urethane 3-2000-1-E. Symbols refer to three

different samples .

so

40

E

c

2 30

O

in

g 20

!

X

LlJ 10

0

SRI Polyether Urethane

3— 1 000/425(70/30) — 1 — X

Fibrinogen, 3.0 mg/ml

0 50 100 150 200 250 300

TIME, min

Figure 2: Extension of human fibrinogen on SRI Polyether

Urethane 3-1000/^25 ( 70/30)-l-X) . Symbols refer

to three different samples.

4
7

CM

cn 5

E

luO 4

z

<

CD

cro q3 ooo

<

2 b

0

Figure 3:

4- 4

4- aD a Q

SRI Polyether Urethane

3-2000-1 -E

Fibrinogen, 3.0 mg/ml

50 i 00 150 200

TIME, min

250 300

Adsorbance of human fibrinogen on SRI Polyether

Urethane 3-2000-1-E. Symbols refer to three

different samples.

6 - CM

cn 5

E

u

O 4

z

<

CD

or o o 3

ooo

<

2

0

Figure 4

+ A

4- .

SRI Polyether Urethane

3-1 000/425(70/30)- 1 -X

Fibrinogen, 3.0 mg/ml

50 100 150 200

TIME, min

250 300

Adsorbance of human fibrinogen on SRI Polyether

Urethane 3-1000/423 (70/30)-l-X. Symbols refer

to three different samples.

5
Ethylcellulose Perfluorobutyrate (Midwest Research Institute)

A one mm thick sheet of sterile ethylcellulose perfluorobutyrate membrane

material was prepared for our ellipsometry studies by Dr. R. J. Peterson (MRI)

,

and received by us on April 27. Although the surface was marred by rippling,

due to the effects of casting such a thick film, sufficient flat portions

were found for use as ellipsometer samples. Preliminary measurements of

fibrinogen adsorption on this material indicate (1) a typical film thickness

of about 30-40 nm and (2) a much greater adsorbance after 2-3 hours than we have observed on any other substrate thus far (20-30 mg/m2 ). There is some evidence that the optical constants of the substrate itself might have been

changing during the course of a given experiment, and further work is in

progress to determine whether the unusually high apparent adsorbance is real

or due to an experimental artifact.

Polyacrylamide Hydrogel (Franklin Institute Research Laboratories)

Eleven individually-prepared sterilized samples of polyacrylamide

hydrogel, grafted with active hydrogen to SRI polyether urethane substrate

(3-2000-1-E) were received from Dr. P. L. Kronick (FIRL) in December 1975.

To date, four samples of this material have been examined by ellipsometry

and only one was found to give the specular reflection which is required to

make the optical measurements. Results from this one sample are ambiguous

and cannot be confirmed without another suitable sample. There are apparently

other differences between the individual samples, as evidenced by (1) the

cracking of the surface on some samples but not others and (2) the ability

to obtain a specular reflection on the "uncoated" side of some samples, but

not others. Given the sample-to-sample variability, we do not feel that it

is meaningful to perform fibrinogen adsorption experiments on these materials,

since the results for any given sample are not necessarily an accurate

representation of the adsorption characteristics of all samples.

Polyacrylamide Hydrogel (Polysciences, Inc.)

Six sterile samples of solution-grafted polyacrylamide hydrogel on SRI

polyether urethane (3-2000-1-E) were received from Dr. 0. Solomon on May 25.

We were unable to obtain a coherent reflection from the surface of these

samples, and therefore could not perform ellipsometric measurements on them.

Examination of a dried sample of this material under the microscope (100X)

revealed that the surface was completely covered with bumps sufficiently

large to scatter light. There were no flat spots to give a specular

reflection. While these results do not directly imply anything about the

biocompatibility of this material, it is clear that protein adsorption

experiments will have to be made by some other means, such as with radiotracer-labeled proteins. The coated substrate also appears to be somewhat

stiffer than the uncoated SRI material, suggesting that some modification

of the bulk mechanical properties of this material has resulted from the

coating process.

6
CURRENT STATUS OF OTHER NHLI CONTRACTOR BIOMATERIALS

Glow Discharge Coatings (Research Triangle Institute)

On June 25, we received from Dr. N. Morosoff (RTI) seven sterile

samples of vacuum-cast polyethylene film, supplied by us, coated by RTI

with an 800 A film of polymer deposited from a glow discharge of tetramethyl disiloxane. We have not as yet examined these samples by ellipsometry

.

Poly alky lsulfone (A. D. Little)

By agreement during a telephone conversation on March 16, 1976, Dr. J. D. Birkett (A. D. Little) agreed to attempt to cast a sheet of polyalkylsulfone membrane material suitable for ellipsometric studies, i.e., at

least 1.5 mm thick. We received such a sample on August 12.

Ion-plated Carbon (General Atomic Corp.)

As we reported in our Annual Report for 1975 (5), we have measured the

refractive index of several samples of "ion-plated" (manufacturer’s

notation) carbon on stainless steel and on glass. Significant differences were found in the indices of different thicknesses of carbon on the same substrate (stainless steel), and in the index of a nominally identical

thickness of carbon on two different substrates (stainless steel and glass).

Because carbon is an absorbing film, we did find that its refractive index,

calculated from its reflection coefficients, becomes independent of the

underlying substrate when the film thickness exceeds about 350 nm. This means that quantitative ellipsometric measurements of protein adsorption on

any carbon-coated substrate should be possible, providing the film is at

least 350 nm thick. According to a letter which we received from Dr. A. Haubold of General Atomic on June 29, 1976, the film thicknesses which

will be used on biomaterials will probably be on the order of 500 nm. Thus,

there should be no problems due to the uncertain index of carbon on these

materials, since the index can be measured independently of the underlying

substrate. We already have a number of 400 nm carbon-coated steel coupons

prepared by General Atomic, and these will be used in future protein

adsorption experiments.

Heparin Bonded to Silastic-coated Glass (Carnegie-Mellon University)

On June 11, we received from Dr. M. S. Morgan (Carnegie-Mellon University)

a number of samples which should provide information about the effect of

heparin on the adsorption of blood proteins. Because of the complexity of

the film on these coated slides, it will be necessary to perform a number

of measurements on partially coated slides, in order to establish the optical

constants of each layer in the film. Dr. Morgan has supplied us with 3 samples each of (1) plain glass slides, (2) silastic-coated glass slides,
(3) slides coated with silastic + the bonding reagent, APTES (y-aminopropyl

triethoxysilane) and (4) all of the above + heparin. Because of the number

of intermediate measurements required by such a complex surface, there may

be considerable uncertainty in the fibrinogen adsorption measurements.

However, we expect to perform such studies on these surfaces and hope that

at least semiquantitative information can be derived.

RADIOTRACER ADSORPTION STUDIES

Effect of Molecular Impurities on Adsorption of HSA

We have been concerned with the use of unpurified proteins for the

measurement of the adsorption of proteins on surfaces. These adsorption

measurements are commonly made using labeled and unlabeled proteins which

may contain varying amounts of dimers and higher aggregates as well as low

molecular weight fragments. Depending upon the relative amounts of these

components and their competitive adsorption properties, the rates and amounts

measured from such mixtures will be different from the desired values of the

monomer alone.

We have analyzed and separated commercially available human serum

albumin (HSA), both labeled and unlabeled, and measured the adsorption of

these fractions on two different substrates, polyethylene and silica. These

fractions were separated on a Sephadex G-200 gel permeation chromatographic

column. Figure 5 shows the results for Ull HSA. Starting from low molecular

weight to high, 31% of the sample is low molecular polypeptide, 49% monomer

and 20% dimer and higher aggregates. In Figure 6 the same separation is

shown for 12^1 labeled HSA obtained from the same commercial source which

gave a similar distribution of products. The relative amounts are also

similar to ^-^I labeled material with 40% polypeptide, 40% monomer and 20%

dimer and higher aggregates. There is no significant difference between the

-*-31i and 125]- sampie<

Because of self- radiolysis caused by the more energetic labeling atom, one might expect that the l J ll sample would have more low molecular weight

fragments than the 125 This analysis does not support that contention.

The lUl HSA was more than four months old, ample time for maximum radiolysis,

but its elution pattern shows no more fragments than the 12^1 HSA, a fairly new sample. The distribution of the radioactivity in the 12^1 sample is

shown in Figure 7. There is no activity in the polypeptide peak, indicating

that it is probably not a radiolysis product, but most likely formed during

the labeling of the material. Therefore, self-radiolysis does not appear

especially significant in determining sample composition.

Following the work presented above, we examined by gel permeation

chromatography a sample of HSA 125]; supplied by another commercial source.

As shown in Figure 8, this material is clearly much less contaminated with

dimer and higher aggregates than that from the other commercial source.

8
Q)

s

3

O

>

c

o oea =2

uj

aoueqjosqv

9

Figure

5

:

Gel

Permeation

Chromatogram

of

I-HSA
0

E

J3

©

>

©

O

+3

3

in

aoueqiosqv

10

125

Figure

6:

Gel

Permeation

Chromatogram

of

I-HSA
monomer

AjjAiiOv

0

E

J3

O

>

c

• oOBB +=>

LJLi

11

125

Figure

7:

Relative

Radioactivity

of

the

I-HSA

Chromatogram

Shown

in

Figure

6.
ABSORBANCE

O

G

O

S

Figure 8: Gel Permeation Chromatogram of

125 I-HSA

12
In Figure 9, the elution curve for the unlabeled HSA is presented.

This sample has less than 0.5% low molecular weight polypeptide, 72%

monom^ and 28% dimer and higher aggregates. For the adsorption measurements,

the “ I labeled material shown in Figure 6 was used and fractions were

collected from each peak.

Table I shows some preliminary results for the adsorption of selected

fractions on polyethylene. The second and third columns are really a test

of internal consistency, experimental reproducibility, and the effect of

the labeling atom. There is good agreement between these two columns in

both amounts and rates. The third column represents an approximation of the

usual practice in adsorption measurements, with only the elimination of

the labeled aggregates. In this case, measurement is made of only the

monomer and the degree to which it is affected by the presence of aggregates.

The adsorbance of the monomer is apparently unaffected by the aggregates at

short times but the values at longer times are distinctly different. The

last column shows the adsorption of the aggregates at two different concentrations. First, at the same concentration (2 mg/ml) as the monomer in

the preceding columns and secondly at a lower concentration (0.2 mg/ml)

corresponding to the concentration of the aggregates in the experiment

reported in the third column. Solutions of the labeled and unlabeled were

made up by weight in pH 7.4 phosphate buffer.

These results for the adsorption of a mixture of monomer and aggregates

(dimer) are shown graphically in Figure 10. In mixtures containing 10%

aggregate, the adsorption of the monomer is shown in Figure 11. The

maximum adsorbance here is lower than that shown in Figure 10 for the monomer

alone indicating that the aggregates can compete effectively with the monomer.

In the second half of Table I, results of similar experiments are shown for the adsorption of HSA on silica at a solution concentration of

2 mg/ml for monomer and 0.2 mg/ml for aggregates. While the total adsorbance

on this substrate is several times lower than that on polyethylene, the

general behavior is quite similar to that observed for the adsorption on polyethylene and the same conclusions would apply. Further experiments are underway to understand and interpret these results more fully.

131 Adsorption Isotherms of HSA I Monomer and Dimer on Polyethylene

Figures 12-16 show the adsorbance, from different concentrations of

HSA '^1 monomer on polyethylene, with readings taken at 0.5, 1, and 3 hour

intervals. Figure 17 is the adsorption isotherm of the monomer, resulting

from the average values of the data presented in Figures 12-16. Similarly,

Figures 18-21 represent the rate of adsorption of the HSA Ji

I dimer on polyethylene and Figure 22 is the adsorption isotherm for the dimer. As

shown in Figures 17 and 22, there seems to be no significant difference

in the total adsorbance between the monomer and dimer.

13
0E3©>c

tj oBE3 +*D

Ui

14

Figure 9:

Gel

Permeation

Chromatogram

of

Unlabeled

HSA
ADSORBANCE

OF

SELECTED

FRACTIONS

OF

125I

LABELED

AND

UNLABELED

HSA

ON

POLYETHYLENE

AND

SILICA

XI 00 co >d0) 03 ON CM 00

4-1 Cl) . • •

cfl 4-> CM 00 o a> d co a rH O 00

•H (1) 0£

4-1

d

a 1—1

ON vO

CJ oo ON 00

cO oo • • • d < <r vD

Cd CM CM CM

oo

co r*-

<}- co

NO 00 NO CO

0- r^.

xl

X

a) a)

4-1 4-1

X

co

X

d co QJ

d

d a> (1)

d i-H 0) CM 00 cr\ NO o >h S X o a) a a CM ON

•H a)

O

d •HX o . • .

4-1 -D

d co 4-1 CO

d 04

O rH o U co o CJ X o a t—H rH rH

CO H-J

a cO 1 a

d

d

d

Cd Pd| d> d

w to 2W < >4 CJ

pr*

M

E—1

X)X rJ

W a) 0)

X

M

>4 4JX

d 4-1 cu

d CO

X co ai a) CO iH 0) CM NO LO 00 CO i—1 O d i—i S X d a) a a NO O co Cs| &4 o a) o d o X o • • . • • •H -Q d CO •H cO d 04 00 o 40 CO

4-1 CO o 4-1 rJ o a rH rH O X a o 1 a co CO d d d X

Pd Pd X

<1)

co

X

d

d CU (U CM o i—! a a

'H 0) o 'r 4J Xl d oc

<J cO o a

COd

Pd

rJ

a

O NO CO

<1- 00

m

ct\ on uo

cua

•HH

C/3 . d

4-1 CO 3 O Q O < X

40 o o • • • rH CO

40 o o • • • rH CO

15

3.95

7.47

4.04
CO

CM

gUj/Sm 'aoNvaaosav

16

TIME,

hrs

125

Figure

10:

Adsorption

of

I

HSA

Fractions

on

Polyethylene.
z iu/6ui goNvaaosav

17

125

Figure

11.

Adsorption

of

1

HSA

Monomer

on

Polyethylene

in

Presence

of

Unlabeled

Aggregates
jiu/Bui aoNvaaosav

18

131

Figure

12:

Adsorption

of

the

Monomer

of

I

HSA

on

Polyethylene.

Solution

concentration

0.309

mg/ml.
ziu/6ui 'goNvaaosav

17

131

Figure

13:

Adsorption

of

the

Monomer

of

I-HSA

on

Polyethylene.

Solution

concentration

0.50

mg/ml.
zui/6ui iDNvaaosav

2C

131

Figure

14:

Adsorption

of

the

Monomer

of

I

HSA

on

Polyethylene.

Solution

concentration

1.30

mg/ml.
jUi/Biu ' 30Nvaaosav

21

131

Figure

15:

Adsorption

of

the

Monomer

of

1

HSA

on

Polyethylene.

Solution

concentration

2.23

mg/ml.
jUj/Buj iONvaaosav

131

Figure

16

Adsorption

of

the

Monomer

of

I

HSA

on

Polyethylene.

Solution

concentration

3.40

mg/ml.
23

Figure

17:

Adsorption

Isotherm

for

the

Monomer

of

I

HSA

on

Polyethylene.
jSu/Bui aoMvaaosav

24

131

Figure

18:

Adsorption

of

the

Dimer

of

I

HSA

on

Polyethylene.

Solution

concentration

0.36

mg/ml.
jUi/Biu 'aoNvaaosav

25

131

Figure

19:

Adsorption

of

the

Dimer

of

I

HSA

on

Polyethylene.

Solution

concentration

0.42

mg/ml.
26

131

Figure

20:

Adsorption

of

the

Dimer

of

I

HSA

on

Polyethylene.

Solution

concentration

0.66

mg/ml.
o © o

r> Lf> co

Z uj/Biu '3DNvaaosav

27

Figure

21:

Adsorption

of

the

Dimer

of

I

HSA

on

Polyethylene.

Solution

concentration

1.30

mg/ml.
jUj/Buj 'iONoaaosav

28

Figure

22:

Adsorption

Isotherm

for

the

Dimer

of

131I

HSA

on

Polyethylene.
FIBRINOGEN ADSORPTION STUDIED BY AUTOMATIC ELLIPSOMETRY

In our Annual Report for 1975, we reported the results of several

adsorption experiments of proteins on platinum, using the automatic recording

ellipsometer. We noted that one of the protein solutions studied, namely

0.42 mg fibrinogen/ml, showed a large increase in the film thickness

(molecular extension) with time. We have recently repeated this experiment

and found the initial thickness of the adsorbed film to be much greater

than that found in the previous experiment. The extension also remained

approximately constant over a time period from two seconds to about two

hours after the initial contact between the solution and the substrate. The

adsorbance increased smoothly and monotonically from 3 to 5 mg/m2 during

the first minute of observation, and from 5 to 7 mg/m2 during the next two

hours

.

Subsequent adsorption experiments were performed from more dilute

solutions (0.043 mg/ml). In one case, the calculated thickness decreased

within the first two minutes. The two hour (equilibrium) value was about

40 nm, which is comparable to values obtained from the more concentrated

solution. The initial adsorbance was lower than that from the more concentrated solution but increased by more than 100% during the first two

minutes and by another factor of two during the next two hours. The final

adsorbance value was also comparable to that obtained from the more concen- trated solution. A second experiment performed at this dilute concentration

proceeded somewhat differently during the first two minutes, but both the

film thickness and adsorbance curves closely paralleled those of the other

experiment during the rest of the 2 hour run. For the first 2 minutes,

the calculated film thickness remained constant at about 50 nm, instead of

apparently decreasing from 120 to 50 nm as in the earlier experiment.

Another adsorption experiment using a very dilute solution of fibrinogen

(0.003 mg/ml) onto a silicon oxide surface proceeded sufficiently slowly

that we could observe all of the adsorption process with the automatic

instrument, beginning about two seconds from the initial contact of the

solution with the surface. The results of this experiment did not indicate

a change in conformation (molecular extension) of the adsorbed protein as the surface concentration increased from 0.05 to 0.75 mg/m2

. However,

other protein-substrate combinations may well produce different results.

All of these results are highly tentative as the reproducibility of

the mixing techniques and the accuracy of the instrument have yet to be

determined, but it is clear that the adsorbance and molecular extension

can be observed and followed, beginning about two seconds from the first

contact of the protein solution with the substrate. We believe that the

instrument is capable of providing valuable information about the rate of

adsorption and conformational changes of adsorbed proteins during the first

critical few minutes after the exposure of a foreign surface to a protein

solution.

29
ADSORBED PROTEIN CONFORMATION BY LIGHT SCATTERING

Quasielastic light scattering techniques have been applied in situ to

study the conformation of y-globulin adsorbed on polystyrene latices. In

collaboration with Dr. Charles Han of the Polymers Division, we have measured

the frequency distribution of scattered light to determine the radius of bare

and protein coated latex particles. Using the basic light scattering equation

Av, = (q

2 /7T)D (1)

"2

where Av;, is the half width of the scattered frequency distribution, q the

scattering vector given by

q

47m

Ao

sin ( 2 )

where n is the index of refraction, Ao the wavelength of light and 0 the

scattering angle, one can determine D, the translational diffusion coefficient.

Combining the Einstein equation for the diffusion coefficient

D = kT/f ( 3 )

where k is Boltzmann's constant, T the temperature, and f the frictional

coefficient, with the Stoke's equation describing the hydrodynamic properties

of a sphere

f = 6 titiR (4)

where n is the viscosity and R the radius of the sphere, one obtains an equation

kT _1 6 Trri D

(5)

which can be used to calculate the radius of a particle from its measured

translational diffusion coefficient.

.

As shown in Figure 23 for the bare polystyrene latex, a plot of HWD(Av^)

vs q

^ / tt results in a linear relation which corresponds to a particle radius

of 44 nm. The adsorption of human y-globulin at 1.04 mg/ml and 0.004 mg/ml

concentration on the latex has been measured. At the higher concentration

where the surface is saturated, the increased particle radius calculated from

30
UJ

31

Figure

23:

Width

at

half

height

(HWD)

of

Scattered

Frequency

Spectrum

vs.

q

2

/Pi

(q

=

scattering

wave

vector)

for

Surfactant

Free

Polystyrene

Latex.
equation (5) is 20 nm, very similar to the ellipsometric extension of

y-globulin adsorbed on polyethylene when a correction for the distribution

of protein segments is made. At the lower concentration where we estimate

about 20% coverage, the increased radius is 15 nm as illustrated in Figure 24.

In order to determine if the observed difference in extension represents

a change in conformation (which would be consistent with our bound fraction

and ellipsometric measurements) , further analysis of the hydrodynamic properties

of partially coated spheres is required. The hydrodynamic measurements in

this case result in an averaging of molecules and "holes" and an underestimation

of the extension. Recent analytical solutions to this problem are being

utilized to assess the magnitude of the effect, and it should be possible to

determine if the measured differences represent conformational changes.

The technique of quasielastic light scattering is applicable to all

materials for which uniform particulate suspensions can be prepared. It has

great precision and can provide additional measures of adsorbed protein

conformations

.

32
o

O CO LD CO O CO LO O CD CD CO CO ID LD

wn 'sni(M Jioiiavd

33

Figure

24:

Radius

of

Polystyrene

Particle

with

Adsorbed

Human

y-Globulin

vs.

Time.
REFERENCES

1.

Batt, C. W. , Mikulka, T. W. , Mann, K. G. , Gaurracino, C. L. , Altiere,

R. J. , Graham, R. G. , Quigley, J. P. , Wolf, J. W. , and Zafonte, C. W.

,

J. Biol. Chem. , 245, 4857 (1970).

2. Laki, K. , Arch. Biochem. Biophys. , J32 , 317 (1951).

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

Stromberg, R. R. , "Interaction of Plasma Proteins with Surfaces," PB

232 629/6 (Available from the National Technical Information Service)

,

Annual Report prepared for the Biomaterials Program, National Heart

and Lung Institute, NIH, Bethesda, Md. , March 1974.

4. Stromberg, R. R. , Morrissey, B. W. , Smith, L. E. , Grant, W. H., and

Fenstermaker, C. A., "Interaction of Blood Proteins with Solid Surfaces,"

PB 241 267/4SL (Available from the National Technical Information

Service) , Annual Report prepared for the Biomaterials Program, National

Heart and Lung Institute, NIH, Bethesda, Md. , January 1975.

5. Stromberg, R. R. , Morrissey, B. W. , Smith, L. E. , Grant, W. H. , and

Dehl, R. E. , "Interaction of Blood Proteins with Solid Surfaces,"

NBSIR 76-1017, Annual Report prepared for the Biomaterials Program,

National Heart and Lung Institute, NIH, Bethesda, Md. , March 1976.

34
APPENDIX

PUBLICATIONS

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

"Conformation of Adsorbed Blood Proteins: Extension and Rate

Studies," Colloid and Surface Chemistry Abstracts, Chicago Meeting

(1973).

2. B. W. Morrissey and R. R. Stromberg, "Conformation of Adsorbed Blood

Proteins," ACS Organic Coatings and Plastics Preprint, _33 (2), 333

(1973).

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

W. H. Grant, "Conformation of Adsorbed Blood Proteins," Proc. of

Symposium of AAMI, New Orleans, La (1974) NBS Spec. Publ. 415.

4. C. A. Fenstermaker, W. H. Grant, B. W. Morrissey, L. E. Smith and R. R. Stromberg, "Interaction of Plasma Proteins with Surfaces," PB 232

629/6 (Available from the National Technical Information Service),

Annual Report prepared for the Biomaterials Program, National Heart

and Lung Institute, NIH, Bethesda, Md. , March 1974.

5. B. W. Morrissey and R. R. Stromberg, "The Conformation of Adsorbed

Blood Proteins by Infrared Bound Fraction Measurements," J. Colloid

and Interface Sci. , 4_6, 1 (1974).

6. R. R. Stromberg, B. W. Morrissey, L. E. Smith, W. H. Grant and C. A. Fenstermaker, "Interaction of Blood Proteins with Solid Surfaces,"

PB 241 267/4SL (Available from the National Technical Information

Service) , Annual Report prepared for the Biomaterials Program,

National Heart and Lung Institute, NIH, Bethesda, Md. , January 1975.

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

"Ellip some trie Investigation of the Effect of Potential on Blood

Protein Conformation and Adsorbance," J. Colloid and Interface Sci.

(in press)

.

8. B. W. Morrissey, C. A. Fenstermaker, "Conformation of Adsorbed y-Globulin

and B-Lactoglobulin. Effect of Surface Concentration," Trans. Amer.

Soc. Artif. Int. Organs (in press).

9. W. H. Grant, L. E. Smith, R. R. Stromberg, "Radiotracer Techniques for

Protein Adsorption Measurements," J. Biomedical Materials Res. (in

press)

.

10. R. R. Stromberg, B. W. Morrissey, L. E. Smith, W. H. Grant and R. E. Dehl,

"Interaction of Blood Proteins with Solid Surfaces," NBSIR 76-1017,

Annual Report prepared for the Biomaterials Program, National Heart

and Lung Institute, NIH, Bethesda, Md., March 1976.

11. B. W. Morrissey, "The Adsorption and Conformation of Plasma ProteinsPhysical Approach," Annals of the New York Academy of Sciences (in press).

USC OMM-NBS L)( 35
'