adsorption of plasma proteins in solution to uncharged, hydrophobic polymer surfaces

J. BIOMED. MATER. RES. VOL. 3, PP. 175-189 (1969)

Adsorption of Plasma Proteins in Solution to Uncharged, Hydrophobic Polymer Surfaces

J. L. BRASH and D. J. LYMAN, Stanford Research Institute, Menlo Park, California 94025

Summary

Infrared internal reflection spectroscopy has been used to study the adsorption of certain plasma proteins on a variety of hydrophobic polymer surfaces. The behavior of the systems studied was almost identical. Under static conditions the proteins appear to be rapidly adsorbed as monomolecular layers from solutions varying in concentration between a few me-% and normal plasma levels. These monolayers are deduced to be closely packed arrays in which the protein molecules appear to retain their native globular form. The bearing of these results on the mechanism of surface-induced coagulation is significant.

INTRODUCTION

It is of prime interest to elucidate the interaction of synthetic polymer surfaces with plasma proteins in solution, since these inter- actions are intimately related to the foreign surface-induced coagulation of blood. Since the intact clotting factors in the cascade scheme of Maefarlane and others1V2 are proteinaceous and have physical properties similar to the more abundant plasma proteins, it is appropriate to draw analogies between the behavior of these two groups of substances. I n particular it would be illuminating to determine whether a given surface is specific in its interaction with a particular protein or clotting factor or whether the interaction is general in the sense of providing an energy source, which can sensitize all or any of the clotting reactions.

Much previous work has been carried out on the behavior of proteins a t s u r f a ~ e s . ~ Most of this has been concerned with the air- water i n t e r f a ~ e , ~ . ~ or the oil-water interface. 3 v 6 Such solid-solution

175

176 J. L. BRASH AND D. J. LYMAN

interfaces as have been studied have been predominantly of the glass t y ~ e . ~ - ~ However there is a need for information on protein behavior a t polymer surfaces. Inasmuch as a relationship has already been established between surface free energy of uncharged, hydrophobic polymers and coagulation time of plasma or blood in contact with them,lO,ll i t would be diagnostic to further demonstrate a progressive specific interaction with some blood constituent such as a protein.

In this paper, therefore, we report on a variety of these uncharged hydrophobic surfaces. These surfaces are uncharged in the sense of having no permanent ionizable groups or semipermanent charges in the manner of electret materials; they will of course have an associated zeta potential in contact with a liquid, due to the electrical double layer. They are also inert in the sense of not containing any reactive chemical groups such as hydroxyl, isocyanate, etc. They represent therefore a relatively simple class of materials whose principal variable property is their surface energy.

Adsorption studies in solution are classically carried out either by depletion methods in which a highly specific surface area powder is exposed to the solution and the decrease in concentration of the latter is measured, or by elution techniques in which the solution and solid are allowed to react, the complex is isolated, and the adsorbed species is washed out and measured. The precision of these methods is dependent on the analytical technique used, and with presently available tools this is usually not limiting. However the intrinsic defect of such methods, especially when dealing Kith biological systems, is that subtle changes such as denaturation at the surface are not readily detected. It is essential to examine the substrate-adsorbate complex directly. Infrared internal reflection spectroscopy appears to provide a technique by which this is possible.

The concept of internal reflection spectroscopy was developed by Fahrenfort12 some years ago, with particular emphasis on the infrared region, and has been reviewed recently by Harrick.13 The technique provides a means of obtaining the infrared spectrum of the first few hundred Angstroms of the surface of a material and therefore could conceivably be used to detect adsorbed layers. Since the infrared spectra of proteins and other biological molecules are distinctive and well documented, the method seemed to have promise for direct study of the protein-surface complex.

ADSORPTION OF PLASMA PROTEINS 177

EXPERIMENTAL

Materials

The polymer films used were: low-density polyethylene (1-mil film, Olin Matheson Corp.) ; polystyrene (5-mil foamed sheet, W. R. Grace Co.)-the foamed material was found more suitable for infrared work than conventional clear film because i t is less brittle and more formable; polydimethyl-siloxane (10-mil medical grade Silastic film, Dow Corning Corp.) ; and fluorinated ethylene-propylene copolymer (Teflon FEP 10-mil film, E. I. du Pont Co.) The films were cleaned by washing in ethanol and then in distilled water. Detergents were avoided since they adsorb and cannot readily be completely removed from the surface of the film.

The proteins used were: albumin (4X crystallized, Nutrional Bio- chemical Co.) ; 7-globulin (Cohn fraction 11, N.B.C.) ; and fibrinogen (Cohn fraction I, N.B.C.). Albumin and yglobulin were found to be electrophoretically homogeneous and were used as received. Fibrinogen was purified by the method of Straughn and Wagner.I4 The Cohn faction was dissolved in a citrate-saline solvent to a concentration of about 300 mg-% and precipitated by the addition of p-alanine. The suspension was equilibrated a t ice temperatures for 30 min and then centrifuged a t 40C for 25 min at 2OOOg. The supernatant was discarded and the precipitate redissolved to a concentration of -900 mg-% and diluted as required.

Adsorption Procedures

The adsorption of plasma proteins to polymer surfaces was conducted under both static and flow conditions. In the static experiments, four sections of polymer film (2 x 5 cm) were immersed in water and protein solution added to make the final correct concentration. Care was taken to ensure that the films did not touch each other or the walls of the beaker. The beaker was covered with foil (to prevent evaporation) and placed in a 37C water bath. After the desired time (usually 2 hr) the solution was displaced by a succes- sion of decantation and dilution steps. The films were then removed from the beakers, shaken to remove large adhering droplets, and rinsed by first immersing in 100 ml of distilled water and then holding in running distilled water for 1 min on each side. Such extensive


rinsing was necessary to ensure removal of all but adsorbed material. The films were then placed in a forced-air oven at 50C and dried for 30 min. The infrared internal reflection spectra were then obtained. In a few instances, water was removed by lyophilization to ascertain that exposure to the 50C drying temperatures had no effect on the adsorbed proteins.

First, the solution (which was contained in a 1 liter beaker in a 37C water bath) was pumped through a loop of tubing of the test material using a tubing pump. Sections of tubing 5 cm long were cut from the loop, rinsed, and dried as in the static experiments; the sections were slit lengthwise and the infrared internal reflection spectra taken. This arrangement was limited to flexible materials available in tube form. The second procedure, using a Babb-Grimsrud artificial kidney test ceU,15 allowed polymers in the form of flat sheets to be studied. I n this arrangement, the polymer films were substituted for the mem- brane. Again a glass beaker served as reservoir and connections were made to the cell with Silastic tubing. Rinsing, drying and spectral measurement were carried out as in the static experiments.

Infrared Internal Reflection Spectra

The spectra were obtained using a Perkin-Elmer 221 spectrophotometer and a single-beam, internal reflection attachment (Model 9, Wilks Scientific Co.). The optical arrangement is shown sche- matically in Figure 1. The reflectance cell used 50 X 20 X 1 mm KRS-5 (thallous bromide-iodide) plates with entrance and exit faces cut a t 45" angles. Spectrometer settings were adjusted to give

In the flow experiments, two procedures were used.

1

SPECTROPHOTOMETER

I

SAMPLE

SOURCE * -_2_

-I

KRS5 REFLECTOR PLATE

Fig. 1. Schematic of optical arrangement for infrared reflection spectroscopy.


the best resolution consistent with a minimum-noise baseline. Ordinate expansion was not necessary except in cases where adsorption was slight. Since conta.ct between film and plate is never complete and can vary for each experiment, it is not possible to utilize the same amount of sample area in each run. However, the intensity of the base polymer spectrum can be used to estimate the relative degree of contact (i.e., area) by determining the ratio of absorbances of the band of interest to a suitable internal standard band in the polymer spectrum. The internal standards used were: the 6.9 p methylene bending vibration for polyethylene and polystyrene; the 7.1 p methyl bending vibration for polydimethylsiloxane; and the 10.3 p -C-F bending vibration for Teflon FEP.

The KRS-5 reflector plate was cleaned in methanol and polished with cerium oxide-chromium oxide (slurried in methanol) between each spectral measurement to remove any protein transferred from the polymer in the preceding run. However, even with this procedure complete removal was not possible and a small but significant layer built up with every spectrum. To compensate for this con- tamination, a control spectrum was run prior to each adsorbed sample using the clean, untreated polymer; the difference between the two absorbance ratios, i.e., the net absorbance ratio, was used in calculating the results. This ratio can, of course, exhibit occasional negative values when adsorption is small and accuracy is reduced.

Calibration of the net absorbance ratio in terms of average surface density of adsorbed material was made by drying measured volumes of solutions of known concentration on a 100 cm2 area of the polymer film and measuring the net absorbance ratio for these known surface densities. This procedure is not suitable for Teflon FEP because its surface is not wetted by water or by any other convenient protein solvent. The spraying of droplets of less than lp diameter (from an atomizer) onto the surface was also ineffective, because they coalesced before drying was complete. Calibration was obtained by a comparative technique, using a previously calibrated surface such as polyethylene on one side of the reflector plate and Teflon FEP on the other side. The relative absorbances of the two internal standard bands (6.9 and 10.3 p ) under identical contact conditions were thereby obtained and were reproducible within a few per cent. Assuming that the extinction coefficient of the amide I band of any protein is independent of the substrate (and this appears to be borne


out by the results), the calibration of the unknown system was obtained by multiplying the protein calibration on polyethylene by the ratio of the internal standard adsorbances, e.g., CFEP = Cp~Aa.g/ Alo.z. The method was substantiated by finding that calibration data obtained directly (where possible) were in good agreement with those obtained by the comparison technique.

RESULTS AND DISCUSSION

The infrared spectra of proteins have been widely studied.16-19 The principal absorption bands of proteins are the amide A band due to the N-H group at 3300 em-' and the amide I and amide I1 bands due to the -CONH- group at 1650 cm-I and 1550 cm-l. These bands are identical for all proteins and peptides, and this prevents distinguishing among different proteins on the basis of their infrared spectra alone. This is illustrated in Figures 2-4, which show the similar infrared reflection spectra obtained for a polymer surface exposed to albumin, fibrinogen, and whole blood, respectively. The latter exposure was made in a flow system,20 which prevents any blood-air interface and thereby any Langmuir-Blodgett transfer. The absorption bands for the proteins adsorbed from blood are indis- tinguishable from those for either albumin or fibrinogen (Figs. 2 and 3)) so it is not possible to say which, if any, of the proteins is preferentially adsorbed.

Since competitive studies cannot be made with this technique, the approach which must be used is that of a comparative study, in which each system is investigated independently but under identical

3 4 5 6 7 8 WAVELENGTH - microns

Fig. 2. Infrared reflection spectrum of human serum albumin on polyethylene: (--) untreated polyethylene; (- - -1 protein treated polyethylene.


0 0

w

a (L 0

2 0.2 m

0.4 a 06 t

7 I .o --_ I co ~ I " ~ ' ' ' ~ ' ~

2 3 4 5 6 7 0 9 WAVELENGTH - microns

Fig. 3. Infrared reflection spectrum of human fibrinogen on Silastic: (-) untreated Silastic; (- - -) protein treated Silsstic.

2 3 4 5 . 6 7 0 9 WAVELENGTH -microns

Fig. 4. Infrared reflection spectrum of polyethylene exposed to whole blood (-) untreated polyethylene; (- - -) whole blood treated polyethylene.

conditions. Using this overall approach, each protein-polymer system was studied by measuring the quantity of protein adsorbed as a function of solution concentration.

How- ever, it was found that buffer salts were adsorbed along with the protein, and that water bound to the buffer could not be removed by any drying procedure that did not change the protein at the same time. Such adsorbed water is sufficient to mask the infrared spectra in the region of interest, i.e., 5-7 p, and cannot be tolerated. It was necessary, therefore, to conduct the adsorption experiments in dis-

Preliminary work was done using buffered saline at pH 7.4.


tilled water. (Several additional experiments were conducted a t pH 7.4 using a,n imidazole-HC1 buffer which does not form water- binding salts. No differences in the results were observed between this solvent and distilled water.)

The quantity of protein adsorbed on each polymer was determined from the ratios of their infrared internal reflection bands. Calibration curves were obtained by depositing known concentrations of protein on the polymer surface. The absorbance ratios were linear with surface density) over the range of interest as shown in Figure 5 for y-globulin on polystyrene. The adsorption behavior of the polyethylene surface toward three of the more abundant plasma proteins, fibrinogen, y-globulin, and albumin) is shown in Figure 6.

The surface concentration of adsorbed protein rapidly reaches a substantially constant value a t solution concentrations of 5-7 mg-yo and does not change even as solution concentration approaches normal plasma levels. At very low solution concentrations, there is a build-up towards this constant) or saturation, value. The scatter

0. I4 rn D z a m 0.12 i o!

- 0.10 (d

0 3 0.08 f 0.06

W ..

W 0

LL 0 rn

0.04 + W z

0.02

0 I I 1 0 0.2 0.4 0.6 0.8 1.0 1.2 1 . f

SURFACE CONCENTRATION - pgcm'' Fig. 5. Calibration of the amide I band in the infrared reflection spectrum of

yglobulin on polystyrene.


SOLUTION CONCENTRATION OF PROTEIN - rng. percent

Fig. 6. Adsorption of plasma proteins to polyethylene a t 37C. ( A ) Fibrinogen; ( 0 ) gamma globulin; (H) albumin.

in the surface concentrations determined a t any given solution concentration reflects the many uncertainties inherent in the method from a quantitative standpoint.

It can also be seen from Figure 6 that each protein has its own definite and distinct surface concentration plateau and that the plateau value increases with molecular weight. It seems reasonable to interpret these plateaus as corresponding to formation of monolayers of protein at the surface. This general behavior has been observed with other types of macromolecules at the solid-solution interface2I and is in general typical of all the protein-polymer systems studies here.

The plateau surface concentrations are given in Table I .

TABLE I Surface Concentration of Plasma Proteins Adsorbed on Polymer Surfaces

Protein conc, pg/cm2

Polymer Albumin 7-Globulin Fibrinogen

Polystyrene Polyethylene Silastic Teflon F E P

0.5 0.7 1.7 0.8 1 .0 1 .3 1.6 1.8 1 . 6 0.8 0 1.4

184 J. L. BRASH AND D. J . LYMAN

It would appear that, within experimental error, the behavior of a given protein is similar for all of these surfaces (e.g., fibrinogen). The most notable exception appears to be r-globulin which has a very high surface concentration on Silastic and zero surface concentration on Teflon FEP. The former result may represent a more compact form of layer on this surface while the latter has no convincing ex- planation a t present. The data did show considerable scatter, and a few experiments indicated finite adsorption of yglobulin on Teflon FEP. Albumin also appears to form an unusually compact layer on Silastic.

The obvious conclusion from these data is that, individually a t least, all proteins behave rather similarly on a wide variety of hydrophobic surfaces. It is tempting, therefore, to infer that in a mixture such as blood the proteins would be adsorbed simply in proportion to their surface collision frequency or concentration. This suggests that albumin would be the most abundant protein found a t the interface, followed by 7-globulin. This idea is contrary to the findings of Vroman who has determined that fibrinogen is preferentially adsorbed at tantalum and silicon surfaces in contact with plasma.22

It was also of interest to determine the effect of flow on the mechanism of protein adsorption. In the in vivo situation, where a pros- thetic surface would be exposed to flowing blood, the accompanying shear stress a t the wall might alter adsorption behavior. For example, adsorption might not occur a t all, or i t may be ac- companied by denaturation, or a continuous adsorption-desorption process may occur with release into the blood of molecules which are denatured and activated as a result of contact with the surface. To study this possibility, we have performed flow experiments on two of the systems studied under static conditions (namely, Silastic- albumin and polyethlene-albumin) . The results are summarized in Table 11. For the Silastic tubing there is no adsorption for up to 12 hr while for Silastic flat sheet a monolayer is almost complete in 15 min, as under static conditions. In polyethylene, which is not amenable to study in tube form, monolayering is complete in 5 min in the flat sheet configuration, again the same as for static conditions.

Thus, adsorption appears to be affected only in the turbulent region. Where flow is laminar, as in the parallel plate configurations, adsorption appears to be the same as for static conditions. I n most areas of the circulation, the critical velocity separating turbulent


TABLE II

Flow Systems Adsorption of Albumin from 20 mg-% Solutions to Polymer Surfaces in

I R net Surface

Surface ml/min number time, min ratio ie/cm2 Flow rate, Reynolds Exposure absorbance cone.,

Silastic: tubing (0.7 cm I.D.)

Silastic : parallel plates (0.29 cm apart

Polyethylene : parallel plates (0.025 cm apart)

Polyethylene

660 2240 240 480 720 960

570 260 15 30 60

240

245 120 5 15 30 60

240

0 1 5

10 15 60

0 0 0.077 0.413

0.066 0.151 0.158 0.200

0.090 0.077 0.071 0.060 0.150

0.110 0.056 0.064 0.081 0.098

0 0 1.0 5 .3

0.86 1.97 2.05 2.60

0.75 1.87

1.4 0.7 0.8 1 .0 1.2

and laminar flow is not exceeded; possible exceptions are the aorta during violent exercise or the regions near heart valves. The study of adsorption of proteins under static conditions should, therefore, have valid application to the circulating blood.

An increase in surface concentration beyond monolayering also occurs a t long exposure times in these flow experiments (see Table 11), for example, after 16 hr in Silastic tubing and after 4 hr in both Silastic and polyethylene flat sheet. This may not be related to flow per se but could reflect some change in the protein which results in adsorption behavior different from that of the native form.

From the surface concentration data in Table I, the protein layer thickness and the average are perprotein molecule can be calculated, assuming protein density to be 1.3 and protein molecular weights of


69 000 for albumin, 160 000 for y-globulin, and 400 000 for fibrinogen. These calculated values are given in Table 111.

If these experimental values are compared to the values calculated from the reported dimensionsz3 of the native globular form of the protein (see Table IV), it should be possible to determine the structure of the adsorbed layer. The experimental layer thickness values in

TABLE I11 Experimental Dimensions of Protein Layers Adsorbed on Polymer Surfaces

Layer thickness, Average area- per Protein A molecule, A2

Albumin 7-Globulin Fibrinogen




on Polystyrene 44 54

130

on Polyethylene 62 77 96

on Silastic 120 138 120

on Teflon F E P 62 0

108

2300 3800 4000

1400 2660 5340

720 1500 4200

1440 0

4760

TABLE IV Dimensional Data for Plasma Proteins

Diameter, Projected area Length, Projected trea Protein ii end-on, A 2 ii side-on, A2

Albumin 40 1700 115 4600 7-Globulin 44 2000 235 10300 Fibrinogen 65 4200 475 13000 to

30000


Table 111 approximate the diameters of the protein molecules, in- dicating a possible side-on adsorption configuration, whereas the area data correspond more to a closely packed layer adsorbed on end.

Since the molecular weight data are probably less questionable than the assumption that the adsorbed protein density is the same as the crystalline protein density, it is felt that the end-on configuration should be favored. If anything, the layer density at the surface would probably be less than that of the crystal and the derived thickness would be correspondingly greater and more in line with end-on adsorption.

Attempts to distinguish between these two configuration may well be unjustified by the accuracy of the data. However, i t does seem clear that the adsorbed material does not consist of a single layer of uncoiled protein. Such a layer which in effect would be a two- dimensional array of amino acids would have a thickness of around 10 A. We conclude then that in general plasma proteins are not drastically denatured in a dimensional sense a t this type of surface, and if initiation of blood coagulation involves adsorption and activation by denaturation, then all of these surfaces should be equally nonthrombogenic, which is not the case.

These data, of course, do not exclude the possibility that the equilibrium layer is a multilayer of denatured protein, that is, suc- cessive layers of uncoiled protein stacked one on top of another. However, if this were the case, i t is difficult to see why the layering should stop a t a definite value greater than one molecule. A more likely alternative is that the native layer could be preceded by a layer of denatured protein next to the surface; this would still be within the limits of the data. However, the layer next to the blood would be a native layer and there would presumably be no progressive denaturation of protein by contact with this native surface.

Protein adsorption in both static and flow systems is not reversible. The proteins are not desorbed over a wide range of pH, nor are they removed by extensive ultrasonic vibration. While this might suggest that the proteins are uncoiled and strongly bonded to the surface through multiple contacts or that a surface-denatured layer-native layer structure exists, such irreversibility has also been observed in the albumin/glass system in which adsorption in the native state was indicated.8

It is of interest to comment on the significance of these results in


regard to the coagulation of blood a t polymer surfaces. It would seem that since factor XI1 and other clotting factors are themselves proteins and are similar to the common plasma proteins in physical properties, then the behavior of these factors a t surfaces should also be similar. Thus factor XI1 is a sialoglycoprotein of molecular weight around 80 000 and migrates between p- and 7-globulin. Furthermore the activated form of factor XI1 behaves as a heavier macromolecule in the ultracentrifuge than does the intact form ac- cording to Donaldson and R a t n ~ f f . ~ ~ The activated form is also less soluble and i t would seem possible that this could be due to some conformational change. If this is so, then activation would be equivalent to a conformational or dimensional change. The con- clusions from these studies, therefore, tend to refute the contact activation of factor XI1 as being the initiating step of clotting on uncharged, hydrophobic polymer surfaces.

The experimental assistance of Miss M. Carini is gratefully acknowledged' This work was supported by the National Heart Institute, Artificial Heart program, under Contract PH 43-64-84, and in part by the National Instititute of Arthritic and Metabolic Diseases, Artificial Kidney Program, under Contract PH 43-66-493.

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17. E. M. Bradbury and A. Elliot, Polymer, 4, 47 (1963). 18. T. Miyazawa, Aspects of Protein Structure, Proe. Symp, Madras, 1963. 19. M. Beer, G. B. B. M. Sutherland, K. N. Tanner, and D. L. Wood, Proc. Roll.

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preparation. 21. A Siberberg, J . Phys. Chem., 66, 1884 (1962). 22. L. Vroman and A. L. Adams, Thromb. Diath. Hemorrhag., 18, 510 (1967). 23. J. L. Oncley, G. Scatchard, and A. Brown, J . Phys. Colloid Chem, 51, 134

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Received October 11, 1968