The adsorption of human plasma albumin and bovine pancreas ribonuclease at negatively charged polystyrene surfaces

The Adsorption of Human Plasma Albumin and Bovine Pancreas Ribonuclease at Negatively Charged Polystyrene Surfaces I. Adsorption Isotherms. Effects of Charge, Ionic Strength, and Temperature W. NORDE AND J. L Y K L E M A Laboratory for Physical and Colloid Chemistry, Agricultural University, De Dreijen 6, Wageningen, The Netherlands Received November 29, 1977; accepted March 3, 1978 This paper describes the adsorption of human plasma albumin (HPA) and bovine pancreas ribonuclease (RNase) on polystyrene latices. The latices are homodisperse and emulsifier-free. Their surface charge is entirely due to sulfate groups. Important variables are the temperature, the pH, the KNOz concentration, and the surface charge on the latex. All isotherms reach welldefined plateaus, but otherwise HPA and RNase behave differently, HPA isotherms sometimes show steps or kinks, their initial slopes increase with temperature (except at low temperature at the isoelectric point), and the plateau values are more or less symmetrical with respect to the isoelectric point. RNase isotherms are of the "high affinity" type, with plateaus that are little sensitive to pH. Interpretation of the data points to an increase in entropy as one of the main driving forces of the process. The pronounced differences in adsorption behavior of HPA and RNase are attributed to the relatively easy adaptability of HPA molecules to changing conditions as compared with the relatively great conformational stability of RNase, so that adsorption reflects the structural coherence of the dissolved proteins. INTRODUCTION

The occurrence of proteins at interfaces is of great biological, medical, and technical significance. In some cases, the interface is a desired locus for the protein (e.g., in biological membranes, digestion of alimentary fats, stabilization of dispersed systems). In other cases, accumulation of proteins at interfaces should be prevented (e.g., adsorption of blood proteins on synthetic body implants as far as this would lead to thrombus formation, or adsorption of salivary proteins on teeth and of marine proteins on ship hulls, where they stimulate growth of microorganisms, etc.). Protein adsorption is also scientifically intriguing. One of the main problems is to what extent conformational changes occur and, if they do, how they modify the biological activity of the protein. Sometimes,

phenomena are observed that are not intuitively anticipated. For instance, a negatively charged protein molecule may adsorb onto a negatively charged adsorbent surface with the evolution of heat. This one example already shows the complexity of the adsorption process. To understand better the various factors playing a role in protein adsorption, measurements with well-defined model systems carried out under well-controlled conditions are indicated. In the present study, we describe the adsorption of human plasma albumin (HPA) and bovine pancreas ribonuclease (RNase) on negatively charged polystyrene latices. The choice of the two proteins has been motivated by the relatively great conformational adaptability of the HPA molecule as compared to the RNase molecule. The latices can be made homodisperse, surfactant-free, with a sur-

257

Journal of Colloid and Interface Science, Vol. 66, No. 2, September1978

0021-9797/78/0662-0257502.00/0 Copyright© 1978by AcademicPress, Inc. All rightsof reproductionin any formreserved.

258

NORDE AND LYKLEMA TABLE I Characteristics of the Polystyrene Latices and the Proteins That Are Used in the Adsorption Studies a

Polystyrene latices (2) Specific surface area Surface area/charged group Surface charge density Electrokinetic potential

(m ~ g-a) (nm 2) ~ C cm -~) (mV)

Electrokinetic surface charge density

(/~C cm -z)

0.05 0.01 0.05 0.01

Proteins Molecular weight Density Shape Size (13, 14) Diffusion coefficient (15, 16) Isoelectric point Conformational stability Hydrophobicity (17)

(g mole -a) (g cm -3) (nm 3) (cm 2 sec -1) (pH units) (kJ/amino acid residue)

M M M M

11.6 7.0 -2.3 -36 -55 -2.0 - 1.5

KNO~ KNO3 KNO~ KNO3

8.9 1.0 -15.5 -52 -75 -3.0 -2.3

HPA (or BPA) b

RNase

69.000 1.35 Ellipsoid 11.6 × 2.7 × 2.7 0.70 × 10-6 4.2-5.0 Low 4.68

13.680 1.42 Ellipsoid 3.8 × 2.8 × 2.2 1.07 × 10-6 ca. 9.2 High 3.57

a T = 25°C. b The properties of HPA and bovine (B)PA are practically identical (see, e.g., Refs. 18 and 19).

face charge o-0 entirely due to sulfate (-OSO~-) groups, variable between ca. - 2 and ca. - 1 5 /xC cm-: 2 (1, 2). In the latter case, roughly a third is covered by hydrophilic groups, whereas for - 2 /zC cm -2 the surface contains so little charge that it is essentially uncharged and hydrophobic. By choosing the pH, i.e., the net charge on the protein as one of the variables, a wide range of electrical affinities between adsorbent and adsorbate is obtained. The nature and concentration of added electrolytes are other important variables controlling the extent of screening of the various charges. Entropic contributions accompanying, for example, the hydrophobic interactions, may be revealed by varying the temperature. In this first paper, we emphasize the adsorption process as such. In subsequent articles (Parts I I - V I of this series), we intend to deal with electrostatic and thermodynamic aspects. Provisional communications have been reported earlier (3, 4, 5, 6). It is hoped that our contribution may help to remove the confusion in this area Journal of Colloid and Interface Science, Vol. 66, No. 2, Septembe r 1978

(7, 8, 9), which is partly due to experimentation with insufficiently controlled systems. MATERIALS

The preparation and characterization of the polystyrene latices have been described previously (1, 2). Electrophoretically pure HPA and RNase [5 × crystallized, salt-free, protease-free, and roughly chromatographed to increase the " A " fraction (10)] were obtained from Sigma Chemical Co. The proteins were dissolved in water and dialyzed for 2 days against a ca. 100-fold excess of water, which was refreshed after 1 day. Without further purification, 1 mole of HPA contains 1 to 2.2 moles of fatty acids (11). Prior to use, the RNase solutions were adjusted to pH 6.5 and heated for 10 min at 62°C to break up possible aggregates (12). All other chemicals used were of analytical grade. The water was distilled from an all-Pyrex apparatus. Some of the properties of the polystyrene

259

ADSORPTION OF PROTEINS I

00=-2.3 laC cm -2

rp

%=-15.5 taC c m -2

% 3O . m g m - 2

3.0 "mg rn-2

T=37°C

T=37°C 2.0

10

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I

o o

I

o~o

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I

I

20 m

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1.0,

I

I

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I

i

I

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,00...4o . - . . . - ~ x 20

-''x-x-x-

I

I

30

l0

T:22°C

,-,-o-O-o,,

x~X~ X,,,X°

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1.0

~o_,~o~o-om

I

c

I

o o

I

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I

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i

I

3.0

IOlO

~

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I

3.0

i

i

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I

i

0 l~ 0

030

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0

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~

1.0 ~o.o.~....oo ~ O ~ O - - o ~ o - - o l o

010

i

~.x~x--

.,.,~ ,.~. o ....,,.-~

o

o

o

o

o

1.0 t

i Cp

0./.,0 0.50g dm -3

I 0.10

I 0.20

I3 O. 0

I 0.40

I 0.50

Cp I 060g dm -3

FIG. 1. Adsorption isotherms for HPA on polystyrene latices: O, pH 4.0; x, pH 4.7; ©, pH 7.0; C~No3 = 0.05 M.

latices and the proteins that may be relevant to the adsorption behavior are summarized in Table I. EXPERIMENTAL

The way of determining adsorption isotherms has been described previously (2, 5). To keep the systems as simple as possible, the use of buffers was avoided. In the systems currently studied, only one electrolyte, i.e., KN03, was added. The experiments were carried out at constant temperature up to the decantation of the supernatant obtained by centrifugation. RESULTS AND DISCUSSION

Examples of isotherms for HPA are given in Fig. 1 and for RNase in Fig. 2. The amount of protein adsorbed, Fp, is plotted

against the protein concentration in solution, cp, after adsorption. The adsorption of HPA and RNase is highly irreversible toward dilution. This is a general feature of polymer adsorption. As a consequence, the amount adsorbed and the conformation of the adsorbed molecule may depend on the conditions under which adsorption occurred. However, the adsorption of both proteins is semireversible toward pH. On changing the pH away from the value where the plateau-adsorption, Fpm, is a maximum, desorption does not occur. Adjusting the pH to the value where Fpm is a maximum leads to additional adsorption up to ca. 80% of the amount that adsorbs in the direct experiment. Apparently, rearrangement of the adsorbed protein layer can occur without molecules being completely desorbed. Journal of Colloid and Interface Science, V o l . 66, N o . 2, S e p t e m b e r 1978

260

NORDE AND LYKLEMA ao:-2.3 I~C c m -2 2.0 -mg m-2

rp

T= 37°C

do =-15.5 IJC c m -2

2.0

mg m-z

T: 37° C

L~e'~-~e'~1.:oIt'%'-&'o'l.'o:%-Jb=-%".to 1.0 ~

0

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I

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tO

I

I

I

I

I

0

2.0

I

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I

I

20 T=22°C

T=22°C ~oq~..eo - C o ,~x~X~X--X

1.0

I

I

I

I

I

0

2.0

I

~ - e - o - e - - o • 4 o .e - o I --X - - X ~ X - - X -- X-~ 9 - - 9 --V - ~ - V - - V - - V - -

I

I

I

I

2.0 T:5°C

T=5°C

1.0 ~ . ~ - ~ . - ~ o5 ~ , , .

1.0

~7,.V--V-V--V-- V--V~7-V

~>.0--0.0. O-- 0--0 ~ 0 ~ 0 - - 0 ---0 ~e X -Dx-oXe X • -x • -x-o -x- o.x- • -X-o ~.V V

v---v

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v

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v

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I

I

0 . 2 0 030

I

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Fie. 2. Adsorptionisothermsfor RNase on polystyrenelatices: @, pH 4.0; ©, pH 7.0; x, pH 9.3; V, pH 11.0; C~No3= 0.05 M. For both proteins, all adsorption isotherms show well-developed plateaus at sufficiently high values of cp, in contrast to most synthetic macromolecules for which the amount adsorbed is often a continually increasing function of the concentration in solution. Unlike the isotherms for RNase, those for HPA exhibit in some cases a definite step or inflection point. This probably reflects a reorientation or possibly a conformational alteration of the adsorbed molecules, rather than the deposition of a second protein layer (5). It seems that the mode of adsorption when the surface concentration corresponds to the initial rising portion of the isotherm differs from that as saturation is approached. For the adsorption of ovalbumin and BPA on various adsorbents, similar steps in the isotherms have been reported (20). These steps were found to correspond with irregularities in the electrophoretic mobilities of the protein-covered substrate particles. Journal of Colloid and Interface Science, Vol. 66, No. 2, September 1978

An adsorption isotherm may be described well by some isotherm equation, but such an interpretation only holds if the premises of the theory are met in the experiment. For this reason, application of Langmuir's theory to protein adsorption would be incorrect. The theories describing polymer adsorption refer to randomly coiled polymers (21, 22) and thus do not hold for globular proteins. To date, no satisfactory theory for protein adsorption has been developed. When discussing the effects of the variables considered on the adsorption behavior, it must be realized that different parts of the isotherm reflect different interactions that may lead to different modes of adsorption. At low coverage of the polystyrene surface, the shape of the isotherm is essentially determined by the proteinpolystyrene interaction. At high coverage, lateral interactions between adsorbed protein molecules may also play a role in the adsorption process. Hence, below, the ini-

ADSORPTION OF PROTEINS I (1o=_2.3gC cm -2

ao :_1551sC cm -2

rp/

B

/

/

mg,,~rrr2 1.2

261

o.,0

! /

J

g ,/~

0.8

pH4.7

,}

f.x

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x

04 0

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

b rng m-2

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pH

7.0

~to

7.0

Jx

Cp I

0

I

0.02

I

I

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I

I

Cp

I

0.06 g d m '3

I

0

0.02

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1

I

I

0.06

I

O . 0 8 g d m -3

FIG. 3. Initial parts of the adsorption isotherms for HPA. Solid lines: CKNO~= 0.05 M; X, 5°C; 0, 22°C; T, 37°C. Dashed lines: CKNO:,= 0.01 M; ©, 22°C. tial and final parts of the isotherms are distinguished.

Effect of Charge All experiments presently discussed have been performed using latices with cr0 = - 2 . 3 and - 1 5 . 5 /*C cm -2, respectively. The charge on the protein molecules is varied by adjusting the p H of the solution. U p o n adsorption of the proteins, a small change of p H is observed. The underlying causes will be discussed in a subsequent

article. The values for the p H of adsorption refer to the situation before adsorption takes place. As a rule, the shapes of the isotherms for RNase are much less sensitive to p H and or0 (i.e., to the overall coulombic interaction) than those for HPA. F o r instance, for cr0 = - 2 . 3 /xC cm -2 the plateau values Fpm for RNase are virtually independent of pH, whereas for H P A a variation up to a factor of 2 is observed. For o'0 = - 1 5 . 5 / x C cm -2, the p H effect is stronger, as may be expected, but both trends continue to be much Journal of Colloid and Interface Science,

Vol.66, No.2, September1978

262

NORDE AND LYKLEMA

more pronounced with HPA than with RNase. All RNase isotherms are of the highaffinity type, whereas HPA isotherms are only so at 37°C. The initial parts of the isotherms and the plateau values are represented in more detail in Figs. 3, 4, and 5, respectively. (Note that because of the high affinity character of the RNase adsorption no initial isotherms are obtainable for this protein.) At a given temperature (except for 37°C) and a given ionic strength, the initial parts of the isotherms for HPA (see Fig. 3) tend to reflect the overall coulombic interaction between the protein and the polystyrene surface. Figure 4 shows that for H P A Fpm(pn) is more or less symmetrical with a maximum around the isoelectric point (iep) of the protein in solution, i.e., pH 4.5 to 4.8. Such trends have been reported for HPA and several other proteins at various interfaces (4, 23-26). It indicates that Fpm is to a large extent determined by the net charge on the dissolved protein molecule. Based on the molecular dimensions of HPA given in Table I and accounting for a hydration layer of 0.5 nm, a closely packed monolayer of unperturbed side-on adsorbed molecules yield a surface concentration of 2.5 mg.m -2. End-on adsorption requires 8.4 mg" m -2. Allowing for an uncertainty in the molecular dimensions and the thickness of the hydration layer of, say, 25%, the maximum value of Fpm may correspond to a complete monolayer of more or less side-on adsorbed, structurally unperturbed molecules. The reduction of Fpm with increasing distance from the iep we ascribe to progressive structural alterations of the adsorbing HPA molecules rather than to the formation of an incomplete monolayer of native molecules (2, 4). This conclusion is consistent with the great structural adaptability of dissolved HPA toward variation in the pH (18, 27). Fpm is a continuous function o f p H , suggesting gradual changes in the protein structure. Journal of CoUoid and Interface Science, Vol. 66, No. 2, September 1978

The influence of pH on Fpm for RNase is much less pronounced. It indicates that only little variation occurs in the mode of adsorption over the pH range studied. Referring to the discussion for HPA, this can be explained by the great structural stability of RNase (14, 28, 29). Only in the case of adsorption on the latex having cr0 = -15.5 /~C cm -2, in 0.01 M KNO3, does Fpm depend markedly on pH. Closely packed monolayers of unperturbed RNase molecules would correspond to adsorbed amounts ranging between 1.2 - 0.3 and 1.9 ___0.5 mg.m -2, depending on whether the molecules are adsorbed sideon or end-on (see Table I). Thus, except at high pH values (i.e., pH ca. 11), the values of Fpm may well represent closely packed monolayers of unperturbed molecules, although the relatively low values obtained for the latex with o-0 = - 2 . 3 / ~ C cm -2 suggest a somewhat different situation. The variation in Fpmfor ~r0 = -15.5/zC cm -2 at 0.01 M KNO3 could perhaps be caused by different degrees of tilting of the adsorbed RNase molecules. Anticipating the discussion on the heats of adsorption in Part V (30), the formation of a second layer in these systems seems to be unlikely. Neither are the RNase adsorption isotherms suggestive of multilayer formation. It is noteworthy that both for HPA and RNase Fpm increases with increasing negative charge at the polystyrene surface, even at a pH greater than the iep, where protein and latex experience an overall electrostatic repulsion. This might be due to an uneven distribution of charge in the protein molecule, causing different degrees of tilting of the molecule at surfaces having different surface charge densities (4). However, the difference in hydrophobicity between the two latex surfaces may equally well be responsible for the observed effect. Hydrophobic interaction between the polystyrene surface and the protein molecule may more or less compensate for the loss of intramolecular hydrophobic interaction of a

ADSORPTION OF PROTEINS I

263

~m m g m -2

3O

•

2.5

20 15 10 05

%

5'o

[

6!0

70

D~ I __

8'0

9o

FIG. 4. P l a t e a u v a l u e s o f a d s o r p t i o n f o r H P A . Solid lines: CKNO, = 0.05 M ; T = 22°C. T , ~ro = - 1 5 . 5 / z C c m - = ; O , (r o = - 2 . 3 / z C c m -2. D a s h e d lines: CKNO, = 0.01 M ; T = 22°C. V , (r0 = - 15.5/zC c m - 2 ; © , (to = - 2 . 3 / z C c m -2. 0 , C~o3 = 0.05 M ; T = 5°C; (r0 = - 1 5 . 5 / z C = 0.05 M ; T = 37°C; o'o = - 1 5 . 5 / z C

c m -2. I I , CKNO3 = 0.05 M ; T = 5°C; (r o = - 2 . 3 / ~ C

c m -2. × , CK~o~ = 0.05 M ; T = 37°C; o"0 = - 2 . 3 / ~ C

protein molecule that unfolds during the adsorption process.

c m -2. + , CKNO,

c m -2.

at a pH greater than the iep. This can best be tested at low surface coverage, where the shape of the isotherm is essentially determined by the protein-polystyrene interaction. For HPA, the variation of the initial parts of the isotherms with increasing CKNO3(Fig. 3) does not fulfill the expectation mentioned above. This indicates that the electrolyte primarily exerts its influence on protein adsorption in a different way, e.g., by affecting the conformational stability of the protein and/or by being adsorbed simultaneously. In Parts III (31) and IV (32), the role of the electrolyte in the adsorption process will be discussed in more detail. Because of the high affinity character of

Effect of Ionic Strength The influence of ionic strength on the adsorption has been studied by determining isotherms (22°C) at two concentrations of KNOz, viz., 0.01 and 0.05 M. In view of the charge-shielding action of electrolytes, it might be expected that as far as the overall electrostatic interaction determines the amount adsorbed, increasing CK~03would lead to a lower affinity between the protein and the polystyrene at a pH less than the iep and to a higher affinity

f•m 2.(3

m g m -2 ~7

v

1.6 i

/

1.2 .

.

• .

.

.

o• .

.

.

.

.

.

.

.

\\ .

.

.

~ v \

_

Q8 NI v O4

-//

I

I

I

5

6

I

I

I

8

9

pH

,'o

FIG. 5. P l a t e a u v a l u e s o f a d s o r p t i o n for R N a s e . S y m b o l s a r e t h e s a m e as t h o s e u s e d in Fig. 4. Journal o f Colloid a n d Interface Science,

Vol. 66, No. 2, September 1978

264

NORDE AND LYKLEMA

the isotherms in RNase adsorption, we are not able to draw conclusions about the influence of CKNO3on the interaction between the protein and the polystyrene surface. The effect of CKNO3 on the maximum amounts of the proteins adsorbed is shown in Figs. 4 and 5. In the isoelectric region of HPA in solution, Fpm is not affected by CKNO3.At other pH values, Fpm increases with increasing C~No3, except at high pH (pH ca. 7), where the effect has disappeared. It has been reported that the conformational stability of PA toward the pH of the medium increases with increasing ionic strength (18, 33). Hence, the larger values for Fpm at higher CKno3could be explained in terms of a reduction in conformational changes in the adsorbing molecules at pH values away from the iep. The maximum amounts of RNase adsorbed on the latex with o'0 = -2.3/~C cm -2 are not significantly influenced by CKNO,.In accordance, the conformational stability of dissolved RNase is relatively insensitive to ionic strength (18). In view of this, the variation of Fpm with CKNO~observed with the latex having O-o = -15.5 /zC cm -2 is probably due to variations in the degree of tilting of the adsorbed molecules rather than to structural alterations.

Effect of Temperature Adsorption measurements were carried out at 5, 22, and 37°C. In the case of RNase, no effect of temperature on the adsorption behavior could be detected. The slopes of the initial isotherms for HPA steepen with increasing temperature, between 5 and 22°C, except at the iep (see Fig. 3). These results point to different adsorption mechanisms at the iep and other pH values. It supports the interpretation of Fpm (pH) in terms of an adsorption with relatively minor structural alterations at the iep and increasing conformational changes Journal of Colloid and Interface Science, Vol. 66, No. 2, September 1978

the further the pH is away from the iep. According to this picture of temperaturedependent conformational changes leading to lower Fpm values, in the isoelectric region Fpm should be essentially the same at 5 and 22°C but reduced upon increasing the temperature to 37°C. Indeed, this is confirmed by the experimental results, as can be seen in Fig. 4. Quantitative interpretation of (8 ln cJ 8T)rp in terms of Clapeyron's law to obtain the enthalpy of adsorption is not justified because the premises of this law are not fulfilled. Nevertheless, the very increase of F, as a function of T, observed over a large range of conditions, is noted. We hope to analyze the thermodynamics of the process more systematically in Part VI of this series (34). CONCLUSIONS The study of the adsorption of HPA and RNase on polystyrene latices reveals several interesting features, many of them difficult to understand. Considering that, at least for HPA, the adsorption mechanism is different at low and at high surface coverage, a transition in mechanism must occur in the intermediate coverage region. The steps or inflection points observed in some of the isotherms for HPA may reflect such transitions. The combined data concerning the effects of pH, CKN03, and temperature on the adsorption isotherms point strongly to a mode of adsorption of the proteins which is largely determined by their properties in solution. More specifically, the differences in adsorption behavior between HPA and RNase can, for a great part, be traced to the fact that RNase does not adapt its structure to environmental changes as readily as HPA. In addition, unfolding of the adsorbing protein molecule is more likely the larger the relative contribution from intramolecular hydrophobic interaction to the stabilization of the molecular structure in solution [cf.

ADSORPTION OF PROTEINS I

Ref. (35)]. For HPA this contribution is larger than for RNase (Table I). From the finding that adsorption still occurs when both the protein and the polystyrene surface are negatively charged and that under these conditions the amount adsorbed even increases with increasing o'0, it is concluded that the overall coulombic interaction does not dominate the adsorption process. Accordingly, the effect of temperature on the adsorption of HPA points to an endothermic process, at least at low surface coverage. Hydrophobic interactions and/or increased rotational freedom (due to a loss of secondary structure) inside the protein molecule may increase the entropy and hence be one of the main driving forces for protein adsorption. ACKNOWLEDGMENTS The authors thank Mrs. E. Eikelboom-Akkerman and Ir. F. C. A. Vekemans for their assistance with determining the adsorption isotherms. The figures are taken from the thesis of W.N., by permission of the Editorial Board of the Communications Agricultural University Wageningen, The Netherlands. REFERENCES 1. Furusawa, K., Norde, W., and Lyklema, J., Kolloid-Z. Z. Polym. 250, 908 (1972). 2. Norde, W., Ph.D. Thesis, Agricultural University Wageningen, The Netherlands, 1976; also Commun. Agric. Univ. Wageningen No. 76-6 (1976). 3. Norde, W., Furusawa, K., and Lyklema, J., in "Sixth International Congress on Surface Active Substances, Z/irich 1972, Proceedings II-1," p. 209. Carl Hanser Verlag, M/Jnchen, 1973. 4. Lyklema, J., and Norde, W., Croat. Chem. Acta 45, 67 (1973). 5. Norde, W., and Lyklema, J., in "International Conference on Colloid and Surface Science", Budapest 1975, Proceedings I. (E. Wolfram, Ed.), p. 205. Akadrmiai Kiad6, Budapest, 1975. 6. Norde, W., and Lyklema, J., "Colston Symposium Papers," in press. 7. James, L. K., and Augenstein, L. G., Advan. Enzymol. 28, 1 (1966). 8. Brash, J. L., and Lyman, D. J., in "The Chemistry of Biosurfaces" (M. Hair, Ed.), p. 177. Dekker, New York, 1971.

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