Kinetics of adsorption of proteins at interfaces: role of protein conformation in diffusional adsorption

Biochimica et Biophysica Acta, 954 (1988) 253-264

253

Elsevier BBA 33137

Kinetics of adsorption of proteins at interfaces: role of protein conformation in diffusional adsorption

Srinivasan Damodaran and Kyung B. Song Protein Chemistry Laboratory, Department of Food Science, Unioersity of Wisconsin-Madison, Madison, 14/1(U.S.A.)

(Received 16 February 1988)

Key words: Protein adsorption; Air/water interface; Protein structure; Serum albumin To elucidate the role of protein conformation in the kinetics of adsorption at interfaces, seven structural intermediates of bovine serum albumin were prepared and their adsorption at the air/water interface was studied. Molecular area calculations indicated two distinct molecular processes, the first being the creation of an area, AA l, for anchoring the molecule during the initial phase of adsorption and the second being the A A 2 cleared during subsequent reorientation and rearrangement of adsorbed molecules at the interface. The AA l values for all the albumin intermediates were the same, indicating that the initial work ~rAA l needed to anchor the molecule at the interface was independent of solution conformation of the protein. Unlike AA i, A A 2 exhibited a bell-shaped relationship with the extent of refolded state of the intermediates. Calculation of diffusion coefficients indicated that greater the unfolded state of the albumin intermediate, the greater was the diffusion coefficient. It is shown that the simple diffusion theory is inadequate to explain quantitatively the kinetics of protein adsorption. Specific, conformation-dependent, solute-solvent and solute-interface interactions also seem to influence the kinetics of adsorption of proteins.

Introduction Adsorption of proteins at fluid interfaces and their behavior in the adsorbed state play an important role in m a n y biological and technological processes, For example, in biological systems where many cellular processes, such as p r o t e i n protein interactions, protein-lipid interactions,

Abbreviations: BSA, bovine serum albumin; ANS, 1anilinonaphthalene 8-sulfonate. Correspondence: S. Damodaran, Protein Chemistry Laboratory, Department of Food Science, University of WisconsinMadison, Babcock Hall, 1605 Linden Drive, Madison, WI 53706, U.S.A.

etc., occur mainly at cell membranes, the protein adsorption at these liquid interfaces plays a crucial role. In technological processes, especially in food and pharmaceutical industries, proteins are often being used as surface-active agents in foamand emulsion-based products. The important step in the formation and stabilization of protein-based foams and emulsions is the initial adsorption of proteins at the interface. Several factors, such as ionic, hydrophobic and conformational characteristics of proteins, affect their kinetics of adsorption at interfaces. To elucidate the influence of protein conformation on adsorption, Graham, Phillips and co-workers [1-5] recently studied the kinetics of adsorption of four structurally different proteins, viz., lysozyme, bovine serum albumin, t-casein and r-casein at

0167-4838/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

254 the air/water interface. The observed differences in the adsorption behaviors of these proteins were attributed to their unique structural properties. For instance, fl-casein has a highly flexible random coil structure and contains no disulfide bonds, whereas lysozyme and serum albumin are highly structured and contain intramolecular disulfide linkages. These differences in the molecular conformations of these proteins are believed to influence directly their rate of adsorption and spreading at interfaces. However, it should be pointed out that differences in the adsorption behavior of those three proteins can not be attributed to conformational differences alone, because differences in their amino-acid composition and distribution will also influence their rate of adsorption. To elucidate the influence of protein conformation alone on the kinetics of adsorption at the air/water interface, we adopted a different approach. We prepared seven structural intermediates of bovine serum albumin and studied the kinetics of adsorption and molecular behavior at the air/water interface. The rationale in this approach is that since the amino-acid composition as well as the amino-acid sequence of these intermediates are not altered, the observed differences in the surface adsorption behavior of these intermediates can be attributed solely to conformational differences. We report that the the surface/ interface diffusion of serum albumin increased with the degree of unfolded state of the molecule, which contradicted the classical diffusion theories. We also provide evidence which suggests that the rate of adsorption of proteins at interfaces is not entirely diffusion-controlled, but is dependent on the subphase potential energy of the molecule and the energy barrier at the surface.

Experimental Crystallized and lyophilized bovine serum albumin (BSA) was purchased from Sigma Chemical Co. (St. Louis, MO). Ultra pure (gold label) N a 2 H P O 4 and N a H 2 P O 4 were obtained from Aldrich Chemical Co. (Milwaukee, WI). All other reagents used in this study were of reagent grade. Double-distilled, deionized, glass-distilled water was used in all experiments.

Refolding of reduced and denatured bovine serum albumin Bovine serum albumin was reduced and denatured as described elsewhere [6,7]. Albumin was dissolved (0.2%) in 0.2 M Tris-HC1 buffer (pH 8.6)/10 M urea/1 mM E D T A / 5 0 mM dithiothreitol. After incubating for 6 h, the pH was adjusted to 4.0. Urea and other small-molecularweight additives were removed by passing the solution through a Sephadex G-25 column preequilibrated with 0.1 M HC1. The tubes containing the protein in 0.1 M HCI were pooled and stored at 4°C. Refolding of the reduced and denatured albumin was carried out as described elsewhere [6]. The rate of refolding of albumin was monitored in terms of regeneration of its capacity to bind ANS with time. The rationale of this approach has been discussed in detail [6]. In a typical experiment, to a known volume of regeneration buffer (0.1 M Tris-HCl (pH 8.0)/1 mM E D T A / 1 . 0 mM reduced glutathione/0.1 mM oxidized glutathione) preincubated at the required temperature, an aliquot of the reduced and denatured albumin stock solution was added such that the final albumin concentration was about 1.0 #M. At intervals of time, 2.5 ml aliquots of the protein solution were withdrawn. To this, 40 /~1 of 0.4 mM ANS solution was added and the fluorescence intensity at 485 nm with excitation at 380 nm was measured in a Perkin Elmer fluorescence spectrophotometer equipped with thermostated cell holder. The fluorescence intensity of the system increased with aliquot sampling time. Since fluorescence emission is due to ANS bound to hydrophobic regions in BSA, the increase in fluorescence with time basically reflected rate of formation of hydrophobic regions as a result of folding from a structureless random state to an ordered state. These initial studies on the refolding behavior of serum albumin were used to determine the optimum conditions for trapping structural intermediates of BSA. To prepare structural intermediates, the reduced and denatured BSA was allowed to refold by adding an aliquot of the denatured protein stock solution to 2 liters of regeneration buffer made up of 0.1 M Tris-HC1 (pH 8.0)/1 mM E D T A / 1 mM reduced glutathion e / 0 . 1 mM oxidized glutathione. The final con-

255 centration of albumin in the regeneration solution was about 1.0 /zM. The structural intermediates were trapped as follows. Aliquots of the regeneration solution were withdrawn at various intervals of time and the refolding was arrested by blocking the free sulfhydryl groups [7,8] by adding iodoacetamide to a final concentration of 0.1 M. The solutions were incubated at room temperature for 30 min, dialysed exhaustively at pH 7.0 against water and lyophilized. Seven structural intermediates of bovine serum albumin were prepared by blocking refolding at 0, 0.5, 1, 2, 3, 6 and 24 h intervals. The disulfide content of these intermediates were determined by the 2-nitro-5-thiosulfobenzoate method [9] as modified [10]. The secondary structure content of the intermediates were analyzed by circular dichroism using a JASCO Model J-41C spectropolarimeter.

Adsorption studies Adsorption of albumin intermediates at the air/water interface was studied by the Wilhelmy plate method [1,11] using a Cahn electrobalance (Model 2000) equipped with a dynamic surface tension accessory (Cahn Instruments Co., CA). A thin platinum plate of 1 cm width suspended from the center of the balance was used as the sensor. In a typical adsorption experiment, protein stock solution (0.1% w / v ) was prepared in 20 mM sodium phosphate buffer (pH 7.0). The solution was centrifuged at 12000 × g for 10 rain to remove insoluble particles. An aliquot (1.0 ml) of the stock solution was diluted to 100 ml with the buffer to obtain a final protein concentration of about 10-3% (w/v). For surface adsorption measurements, 90 ml of the diluted sample was poured gently into the Teflon-coated Langmuir trough with the platinum sensor plate hanging in position. The surface area of the trough was 71 cm z and the solution in the trough was 1.2 cm deep. The trough was placed in a water bath which was maintained at 25 + 0.2 ° C. The surface was cleaned by gently sweeping with a fine capillary attached to an aspirator until the surface tension was equal to that of the buffer (72 m N / m ) . The protein was then allowed to adsorb from the unstirred subphase to the air/water interface. The change in

surface pressure, rr, was recorded continuously on a strip chart recorder at various chart speeds. Spread monolayer studies on the albumin intermediates were done according to methods described elsewhere [12,13]. In these experiments, the buffer was taken initially in the trough with the platinum plate hanging in place. The Teflon barriers were lowered into the trough and moved to maximum distance from the center of the trough. A thin glass rod, clamped to a jack, was dipped Up to 5 mm into the subphase. An aliquot (50/zl) of albumin solution containing 10/zg protein was slowly run down onto the glass rod using a syringe. The rod was then gently removed from the surface. The film was allowed to equilibrate until there was no change in surface pressure with time. The film was then compressed stepwise and allowed to equilibrate until there was no change in surface pressure. For each area of the film the equilibrium surface pressure was measured. Results and Discussion

Conformational characteristics of the intermediates The rate of increase of ANS fluorescence in a typical refolding experiment is shown in Fig. 1. The time-dependent increase in fluorescence intensity reflects the rate of formation of hydrophobic regions in BSA as a result of folding from a structureless random coil to an ordered folded structure. Since the formation of hydrophobic regions in proteins is related to the three-dimensional surface or topography of the protein, and not necessarily to the formation of the secondary structures per se, the data in Fig. 1 basically reflect the rate of formation of tertiary structure of BSA. The ordinate in Fig. 1 is expressed in the form of %Ft/Fn, where F t is the fluorescence intensity at time t during the refolding process and F n is the fluorescence intensity expected if the protein completely regains its native structure. The F n value was determined by interacting ANS with native BSA under identical ANS and BSA concentrations as in the refolding experiment. Thus, the ordinate in Fig. 1 indicates percentage regain of native tertiary structure of BSA as a function of refolding time. It should be noted that on the basis of ANS binding capacity, the maximum regain of native structure is only about 30% after 24 h.

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i n t e r m e d i a t e 3, the C D spectra of all other intermediates exhibited negative m i n i m a at 207 a n d 221 nm, which are indicative of a-helical structure [14]. The t i m e - d e p e n d e n t increase in the negative ellipticity at 221 n m a p p a r e n t l y reflects increase in the a-helical c o n t e n t d u r i n g the refolding process. A detailed analysis of the secondary structure c o n t e n t of these intermediates a n d the refolding p a t h w a y of BSA will be presented elsewhere. However, for the present study, the percent increase in [01221 recovery (Table I) will be used as a measure of the extent of secondary structure form a t i o n in these i n t e r m e d i a t e s [7]. The percentage regain of A N S b i n d i n g capacity (Fig. 1, T a b l e I) will be t a k e n as the extent of regain of the native tertiary structure of BSA.

T i m e (hr)

Fig. 1. Rate of refolding of reduced and denatured bovine serum albumin at 25 °C as measured by the rate of regeneration of ANS binding capacity. The regeneration medium was made up of 0.1 M Tris-HC1 buffer (pH 8.0)/1 mM EDTA/0.1 mM oxidized glutathione/1.0 mM reduced glutathione. Albumin and ANS concentrations were 0.33 M and 7.2 M, respectively. The ANS fluorescence was measured at 485 nm with excitation at 380 nm. Ft is the fluorescence intensity at time t, and Fn is the fluorescence expected if the molecule completely refolds to the 'native' structure.

O n the basis of the data in Fig. 1, seven structural intermediates of BSA were prepared b y blocking the refolding process at 0, 0.5, 1, 2, 3, 6 a n d 24 h time intervals. The circular dichroic (CD) spectra of these intermediates are shown in Fig. 2 a n d some molecular properties of these intermediates are given in T a b l e I. Except for

Adsorption at the a i r / w a t e r interface The time-course of changes in surface pressure of a l b u m i n solutions over a period of 15 h are shown in Fig. 3. Except for the intermediates 1 a n d 2, the s u b p h a s e p r o t e i n c o n c e n t r a t i o n in all cases was a b o u t 1 . 0 . 1 0 - 3 % ( w / v ) . Because the solubilities of the i n t e r m e d i a t e s 1 a n d 2 were poor, a d s o r p t i o n studies for these intermediates were c o n d u c t e d at s u b p h a s e c o n c e n t r a t i o n s 0.3 • 1 0 - 3% a n d 0 . 5 5 . 1 0 - 3 % , respectively. I n the cases of a l b u m i n intermediates, the steady-state ~r values were a t t a i n e d w i t h i n 2 h, whereas the native alb u m i n required a b o u t 15 h. Both the rate of increase of ~r a n d the steady-state ~r values for the intermediates were significantly greater t h a n those of native a l b u m i n (Fig. 3). A m o n g the intermediates, the steady-state ~r values a p p a r e n t l y

TABLE I STRUCTURAL PROPERTIES OF BOVINE SERUM ALBUMIN INTERMEDIATES Intermediate 1 2 3 4 5 6 7 Native

Refolding Time (h)

[ 0 ] 221

% [ 0] 22]

% Regain of

N u m b e r of S - S

(deg. cmI. dmol - 2)

recovery

native structure a

bonds regained

0 0.5 1.0 2.0 3.0 6.0 24.0 -

- 7693 - 10496 -6100 - 15 300 - 19160 - 20090 - 20410 - 21220

36.0 49.5 28.7 49.5 72.0 90.0 94.5 100.0

6.5 9.3 13.1 20.7 24.4 25.0 30.0 100.0

0 6.5 8.5 12.7 13.3 13.7 15.2 17.0

a These are Ft/F~ values from Fig. 1.

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Wavelength (nrn) Fig. 2. Circular dichroic spectra of BSA intermediates. Spectra were obtained in 20 m M sodium phosphate buffer (pH 7.0). The path-length was 1 m m and the protein concentration was 0.001 to 0.01%. Intermediates: ©, 1; t,, 2; o , 3; t2, 4; o, 5; A, 6; II, 7. ~ , Native BSA.

decreased with the extent of refolding of the protein. For example, at comparable subphase concentrations, the steady-state ~r values of intermediates 3-7 decreased in the order 3 > 4 > 6 > 5 > 7 > native. It should be noted that, although the % [01221 recoveries of intermediates 6 and 7 were almost the same and nearly equal to that of the native BSA, the steady-state ~r values for these three intermediates differed very significantly (Fig. 3). This suggests that even though the a-hehcal content of these molecules may probably be the same, the tertiary structures, i.e., the way in which the secondary structure segments are arranged in the three-dimensional space, of these intermediates are significantly different. This seems to affect the rate of adsorption, the degree of equilibrium spreading, orientation of peptide segments and the molecular area covered by the protein at the interface. These in turn affect the surface pressure of these protein films at the air/water interface. In order to obtain some insight into the role of solution conformation of BSA on its mechanism

of adsorption and subsequent behavior of the adsorbed molecules at the interface, the ~r vs. t data were analysed further. The rate of adsorption of amphiphiles at interfaces is given by [15,16]: d F / dt = K C o exp( - ~rAA / k T )

(1)

where K is the rate constant of adsorption, CO is the bulk concentration, ~r is surface pressure, A A is the area to be cleared for adsorption, k is the Boltzman constant and T is the temperature. The basic premise in this approach is that for a protein molecule to clear and occupy an area AA at the interface against the surface pressure ~r, it should possess the molecular energy equal to or greater than ~rAA. Since d F / d t = (d~z/dt)(dF/d~r), Eqn. 1 can be expressed as [16]: ln(drr/dt) = I

KCo

n (dF/d~r)

~rAA kT

(2)

To understand the mechanism of adsorption of BSA intermediates, the ~r vs. t data were analyzed using Eqn. 2. The d~r/dt at each ~r value was

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Timo Fig. 3. Variation of surface pressure with time during adsorption of albumin intermediates at the air/water interface. The substrate was 20 mM sodium phosphate buffer (pH 7.0). The bulk concentrations of various intermediates were: o, 1, 0.308.10-3%; zx, 2, 0.559-10-3%; o , 3, 0.95.10-3%; D, 4, 0.99.10-3%; O, 5, 0.996.10-3%; A, 6,0.922.10-3%; II, 7, 0.973.10-3%; @, native BSA, 0.834.10- 3 %.

calculated from the data in Fig. 3 using a nonlinear curve-fitting procedure. The ln(dTr/dt) vs. rr plots for all the intermediates and native BSA were nonlinear (Fig. 4). However, the nonlinear curves could be divided primarily into two regions. The first linear region for all the albumin samples occurred in the range of 3-12 m N / m surface pressure. The second linear region was observed in the 14-24 m N / m surface pressure range. This suggests that at least two molecular processes are involved during formation of protein films at the air/water interface. It has been suggested [1] that the first phase which occurs in the range of 3-12 mN/m may be attributed to initial penetration and anchoring of the molecule at the interface. The second phase, which occurs near the saturation region of the ~r-t curves, may correspond to rearrangement and reorientation of the adsorbed molecules at the interface [1]. Two AA values, i.e., AA 1 and AA2, were obtained from the slopes of the first and second linear regions in Fig. 4. These values are presented in Table II. The AA 1 values correspond to the initial areas cleared by the protein molecules to anchor themselves at the interface and the AA 2

values are the areas cleared during subsequent rearrangement and reorientation at the interface. It should be noted that, with the exception of the intermediates 1 and 3, the AA 1 values of all the intermediates and the native BSA were almost the same, i.e., about 60 ~2. This value is in good agreement with 50 + 8 ~2 reported by Graham and Phillips [1]. Assuming that the average surface area of an amino-acid residue is about 15 ~2 [1,17], it can be predicted that a peptide segment TABLE II S U R F A C E AREAS C L E A R E D Surface area cleared per protein molecule during initial adsorption (AA1) and subsequent rearrangement (AA2) at the air/water interface. Intermediate

AA 1 (,~2)

AA 2 (~2)

1 2 4 5 6 7 Native

135.4 52.6 48.9 64.7 56.8 60.5 77.5

212.7 161.0 297.3 534.0 401.6 259.2 204.5

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Surface Pressure (raN/m) Fig. 4. The ln(d~r/dt) versus ~r plots for albumin intermediates. The d~r/dt values at each ~r value were obtained from the data in Fig. 3 using a nonlinear curve fitting procedure. Intermediates: o, 1; z~, 2; 12, 4; I, 5; A, 6; I, 7; ~, native BSA.

containing about 3 to 5 amino-acid residues is involved in the initial penetration and anchoring of the protein molecule at the interface. The fact that the AA 1 values for the native as well as all the other structural intermediates are the same implies that the initial work, 7rAA] (in the range 3 - 1 2 m N / m surface pressure), needed to penetrate and become anchored to the interface is independent of solution c o n f o r m a t i o n of the protein. Previously, it has been pointed out that, for various structurally very different proteins, the AA values were within the range of about 100-175 A2 and exhibited no dependence on either the molecular shape or size [17]. This analysis is in accord with our results. However, it should be noted that, while the AA] is not dependent on the solution c o n f o r m a t i o n of the protein, the rate of adsorption is very m u c h dependent on protein conformation: the higher the unfolded state of the protein, the greater is its rate of adsorption (Fig. 3). As a corollary, it can be stated that the rate of adsorption is not limited by the energy barrier, ~rAAa, but is very m u c h influenced by the conformational state of the protein in solution. Unlike AA1, the AA 2 values, i.e., the interfacial areas cleared during rearrangement and reorientation of the adsorbed molecules, were dependent on the solution c o n f o r m a t i o n of the intermediates

(Table II; Fig. 5). The AA 2 values progressively increased with the extent of formation of a-helical structure and reached a m a x i m u m value at about 80% recovery of [01221; any further increase in

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Fig. 5. Relationship of z~A] (surface area cleared dunng initial adsorption) and AA2 (surface area cleared during rearrangement) with % recovery of mean residue ellipticity at 221 nm of the intermediates. Filled symbols indicate corresponding values for native BSA.

260

a-helical content and folding of the protein decreased the AIA2 value. In other words, the results tentatively suggest that the unique folded structure, that is, both the secondary and tertiary conformations of the intermediate 5, seems to possess the ability to occupy greater surface area. However, it should also be realized that this ability per se does not ensure greater surface activity, because the equilibrium surface pressure is lower than in the intermediates 1-4 (Fig. 3). It is generally considered that, because of high flexibility and lack of intramolecular constraints, a highly unfolded protein would occupy a greater area at the interface [1,2]. However, the results presented in Fig. 5 apparently do not support this view, because the intermediate 1, which is highly unfolded and contains no disulfide bonds, actually occupied less surface area (AA2) than the other folded conformations. In fact, the data suggest that, for a protein to occupy a greater surface area, the protein should contain a partially folded structure. To confirm this observation, we studied the properties of spread protein monolayers of the albumin intermediates. The relationship between surface pressure and surface concentration of

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spread monolayers is shown in Fig. 6. All intermediates exhibited an S-shaped relationship. However, it should be noted that the surface concentration required to exert a given surface pressure was the highest for intermediate 1 and lowest for intermediate 5. Since 1/F is the surface area occupied per mg protein, intermediate 1 apparently occupied less surface area than intermediate 5. The inset of Fig. 6 shows the relationship between 1/F and % recovery of [81221 at 10 m N / m surface pressure. It can be seen that, as in the case of adsorbed film (Fig. 5), the intermediate 5 occupies the maximum molecular area at the air/water interface. Furthermore, the shapes of the curves in Figs. 5 and 6 (inset) are strikingly similar. These results strongly suggest that neither the completely unfolded nor the compact folded native albumin has the ability to occupy large area at the air/water interface. Apparently, an optimum degree of folded conformation seems to be essential to occupy a greater area at the interface. However, even though the area occupied per molecule of intermediates 1 - 4 is less than that of intermediate 5, at comparable subphase concentrations, both the rate of change of surface

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degree of folding. For example, at 1 • 10-3% subphase concentration, the amount of BSA adsorbed at equilibrium conditions (15 h) was about 7 m g / m 2 for the intermediate 3 as compared to about 2 m g / m 2 for native BSA.

o

Diffusion coefficients of BSA intermediates It is generally accepted that the rate of arrival of protein molecules at the interface is diffusion controlled [1,16,19] and is given by [20]:

¢0

o o

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d F / d t = C0(Ds/3.1416 t) 1/2

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Surface concentration (mg/m2)

Fig. 7. Comparison of ~r vs. F relationship of 'spread' and 'adsorbed' monolayers of native BSA: o o, spread monolayer (this study); e, adsorbed monolayer (taken from Fig. 2 of Ref. 2).

pressure as well as the extent of surface pressure at equilibrium are greater for intermediates 1 - 4 than for 5. This could be due to the adsorption of a greater number of molecules (intermediates 1-4) at the interface. The striking similarities of the curves in Fig. 6 (inset) and Fig. 5 (AA2) strongly suggest that the surface behavior of albumin intermediates in the 'adsorbed' and 'spread' monolayers is very similar. To confirm this, we compared our 7r vs. F curve of the spread monolayer with that of adsorbed monolayer studies of Graham and Phillips [2]. (Fig. 7). The data shown in Fig. 7 clearly indicate that the behavior of BSA in the 'adsorbed' and 'spread' films is indeed very similar. Based on this evidence, using the rr-F curves of spread monolayers (Fig. 6) it was possible to transform the ~r-t curves (Fig. 3) into F-t curves. A similar approach has been used in the case of adsorption of apolipoprotein A-I at the air-water interface [18]. The F vs. t curves for albumin intermediates are shown in Fig. 8. The rates of adsorption of albumin intermediates are significantly greater than that of native albumin. Furthermore, the data indicate that both the rate and the extent of adsorption of albumin apparently decreased with

(3)

where CO is the bulk concentration and D s is the calculated diffusion coefficient. However, many experimental studies on protein adsorption at the air/water interface have shown that the calculated diffusion coefficient has always been lower than the conventional diffusion coefficient in solution [15,16,19]. It has been suggested that the decrease in the rate of adsorption below the rate of diffusion might be due to an energy barrier at the interface, which is related to the energy needed to clear an area A A against the surface qr in order for the molecule to adsorb [15,20]. In other words, the rate of adsorption is given by: d F / d t = Co(Do/3.1416 t) 1/2 exp( - IrA A / k T )

(4)

where D o is the conventional diffusion coefficient in solution. Combining Eqns. 3 and 4, it follows that: (Ds) 1/2 = (Do) 1/2 exp( - I r A A / k T )

(5)

To understand the role of protein conformation on its diffusion to the interface, the adsorption curves in Fig. 8 were analyzed according to the integrated form of Eqn. 3 [19,20]: Ft = 2C 0 (Dst/3.1416) 1/2

(6)

Plots of LP vs. t 1/2 were linear for all the intermediates in the interval between 1 and 10 min of the initial phase of adsorption. Using this short time approximation, the diffusion coefficients of the BSA intermediates were calculated from the slopes. These values are given in Table III and the relationship between the diffusion coefficient and

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Fig. 8. Rate of increase of surface concentration during adsorption of BSA intermediates at the air/water interface. The data were generated by transforming ~r-t curves of Fig. 3 using the ¢r-Fcurves of Fig. 6. Conditions and keys to the symbols are the same as described in Fig. 3.

the percentage regain of native structure is shown in Fig. 9. The calculated diffusion coefficient, Ds, increased dramatically with the extent of unfolding of the protein structure. For example, the calculated diffusion coefficient of native BSA was about 0.18-10 -7 cm2/s, whereas that of the unfolded intermediate 1 was about 24.5 • 10 -7 cm2/s, which amounts to an approx. 136-fold difference. Careful analysis of the results reveals certain discrepancies in the simple diffusion theory of

TABLE III C A L C U L A T E D D I F F U S I O N C O E F F I C I E N T S (Ds) O F BOVINE S E R U M A L B U M I N I N T E R M E D I A T E S Intermediate

% Refolding

D s ( >( 107)

(cm2/s) 1 2 3 4 5 6 7 Native

6.5 9.3 13.1 20.7 24.4 25.0 30.0 100.0

24.50 9.23 8.82 5.68 1.89 1.56 0.80 0.18

protein adsorption. First of all, the observed diffusion coefficient, D 0, of BSA in aqueous solution is about 5.94.10 7 cm2/s [21], whereas the diffusion coefficient calculated from the adsorption experiment is about 33-fold smaller. Moreover, the calculated diffusion coefficient, D s, increases dramatically with progressive unfolding of the protein. This, on the basis of classical diffusion theory, is unreasonable. Since the frictional coefficient of a protein in the unfolded state would be greater than that in the folded state, from the Stokes-Einstein equation, Ds = kT/f, one would expect a decrease in D s with progressive unfolding of the molecule. However, the diffusion coefficients of the intermediates calculated from adsorption data actually exhibit the opposite trend. Although very unlikely, it is possible that some of the intermediates might exist in the aggregate form in solution, which might affect their rate of adsorption. However, since the diffusion coefficient is inversely proportional to the cube root of the molecular weight, the aggregates should exhibit lower, rather than higher, diffusion coefficient. In the light of these arguments, the data strongly suggest that the simple diffusion theory cannot

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% Regain of n a t i v e s t r u c t u r e

Fig. 9. Relationship between logarithm of diffusion coefficient and % regain of native structure (measured as % regain of ANS binding capacity) of BSA intermediates.

adequately explain the process of protein adsorption at fluid interfaces. In order to explain the deviation of the calculated diffusion coefficient, Ds, from the conventional Do, Ward and Tordai [20] originally proposed the idea that an energy barrier might exist at the surface for adsorption. According to this proposal, in cases where the calculated diffusion coefficient is much smaller than the conventional D o, it is not the diffusion but the energy barrier which plays the controlling role in adsorption. To test this, we analysed Eqn. 5 to obtain the predicted Ds value for native BSA by substituting D o = 5 . 9 4 - 1 0 - 7 c m 2 / s , ~ = 1 m N / m , AA = 60 ,~2 and T = 298 K. The predicted D s for native BSA is 4.45.10 -7 cm2/s, whereas the actual D s, 0.18. 10 -7 cm2/s, is about 25-fold smaller than the predicted value. This tentatively suggests that the energy barrier theory alone cannot quantitatively account for the deviation of D~ from D 0. Furthermore, it should be pointed out that any energy barrier theory, whether it originates from interfacial energy barrier or electrical potential energy barrier [15], will be able to explain the reason for smaller D s values only, but will fail to account either for the progressive increase in Ds with unfolded state of BSA or the situation where the

D s is greater than D 0. For example, the calculated D s for the intermediate 1 is much greater than either the calculated conventional D o value of the native BSA. This, on the basis of the energy barrier theory, would mean existence of a negative, instead of positive, energy barrier at the surface for adsorption of intermediate . This is unreasonable. The kinetics of adsorption of proteins at interfaces is highly complex and poorly understood. The data presented here tentatively suggest that the kinetics of adsorption of a protein are governed not only by diffusion and the energy barrier at the interface, but also by its conformational state in the subphase. It is very difficult to relate the rate of adsorption quantitatively to the conformational states. However, in qualitative terms the differences in the rates of adsorption of albumin intermediates may be explained as follows. Adsorption of proteins at interfaces is usually considered to be an irreversible process; however, when there is an energy barrier to adsorption, the net rate of arrival of the protein at the interface is a function of its kinetics of adsorption and desorption [1]. That is: d F / d t = klC o e x p ( - ~ r A A / k T ) - k2F e x p ( - r r A A / k T )

(7)

where k x and k 2 are first-order rate constants for adsorption and desorption, respectively. The observed differences in the rates of adsorption of the albumin intermediates may arise from the relative magnitude of differences in k I and k 2. In the highly unfolded state, because of the extensive binding of the protein segments to the interface, the desorption rate of the protein may be much smaller than that of the folded state. This may cause an apparent increase in the net rate of adsorption of the unfolded protein. Although this simplistic reasoning seems to be adequate to explain the data qualitatively, it is probable that other complex solute-solvent interactions may be involved, which may have greater influence on the structure-dependent adsorption behavior of albumin intermediates. In order to better understand the role of protein conformation in the kinetics of protein adsorption at interfaces, further systematic studies involving other model proteins are needed.

264

Acknowledgement T h i s r e s e a r c h was s u p p o r t e d in p a r t b y t h e College of Agricultural and Life Sciences and by National Science Foundation Grant No. CBT8616970.

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