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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 V. Microcalorimetry 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 As a further means to unravel the various factors involved in protein adsorption, this paper deals with the determination and the discussion of the enthalpy change resulting from the adsorption of human plasma albumin and bovine pancreas ribonuclease on polystyrene latices. The measurements were made under conditions of plateau-adsorption at various temperatures, electrolyte concentrations, and electrical charges on the adsorbate and the adsorbent. Several factors contribute to the observed enthalpy effect. For example, at high pH, where the protein and the polystyrene surface repel each other electrostatically, spontaneous exothermal adsorption takes place. However, under many other conditions the adsorption proceeds endothermically. This and other considerations lead to the conclusion that the adsorption is, at least in part, entropically driven.
INTRODUCTION
The variation upon adsorption of the thermodynamic characteristic functions constitutes an important additional piece of information for a better understanding of the properties of proteins at interfaces. In Part I (1) of this series the irreversibility of the adsorption of human plasma albumin (HPA) and bovine pancreas ribonuclease (RNase) at polystyrene surfaces has been stressed. Because of this irreversibility, it is not valid to derive from adsorption isotherms a binding constant from which the Gibbs free energy of adsorption AadsG can be calculated. For this reason and also because in the case of protein adsorption isosteric conditions defer unambiguous definition, the physical meaning of the adsorption enthalpy calculated from the temperature dependence of the isotherms (according to Clausius-Clapeyron) is questionable. However, since the interracial
area on which, in a latex, adsorption takes place is high, the net enthalpy AadsH due to the overall adsorption process can be determined by calorimetry. For polymer adsorption only a limited number of such determinations have been reported in the literature. Killmann and Eckart (2) have established enthalpies of adsorption of polyethylene glycol on silica surfaces. Calorimetrically they found negative values, whereas the enthalpy evaluated according to the Clausius-Clapeyron equation appeared to be positive. In other words, for polymer adsorption the phenomenological isosteric heat is not a good measure for AadsH , probably because in this case it is not sufficient to characterize "isosteric" as "at constant amount adsorbed." Calorimetric determinations of AadsH for y-globulin on glass surfaces have been made by Nyilas et al. (3). For these systems AadsH per mole of protein adsorbed
295 0021-9797/78/0662-0295502.00/0 Journal of Colloid and Interface Science, Vol. 66, No. 2, September 1978
Copyright © 1978 by Academic Press,~Inc. All fights of reproduction in any form reserved.
296
NORDE AND LYKLEMA
is maximum at the surface concentration corresponding to the completion of the first layer, and it shows a sharp decay on further increasing the amount adsorbed. The authors suggest that the y-globulin molecules adsorbed in the first layer undergo the largest structural changes that could be triggered by the large adsorption enthalpy. We are inclined to interpret A a d s H in a different way. It has to be realized that AaosH is the compounded enthalpy change due to all factors, including structural alterations in the protein molecules that are involved in the overall adsorption process (1, 4-7). For some of these factors the contribution to haasH may be estimated. If the overall AadsH is experimentally obtainable, subtraction leads to the contributions to AadsH of otherwise inaccessible factors. In doing so, more information can be gained on the forces responsible for protein adsorption. This will be discussed in Part VI (8). Anticipating this treatment, we present now the results of direct microcalorimetry. In studying the adsorption mechanism it is informative to determine AadsH at various temperatures. The temperature coefficient (at constant pressure) of AadsH equals the change upon adsorption of the apparent heat capacity 6Cp of the system Aads6Cp -- { S A a d s H / S T } p .
[1]
The quantity Aads~'Cv is a measure of the structural changes that the protein molecule and the other molecules involved, i.e., water and electrolyte, undergo during the adsorption process. The experimental data presented in this paper refer to conditions of plateau-adsorption (1). The variables studied are the pH of adsorption, the surface charge tr0 of the polystyrene latex particles, the concentration CKNO,of KNO3 in solution, and the temperature T. MATERIALS
For a description of the polystyrene latices and the proteins we refer to Refs. (1)
and (7). The other chemicals were of analytical grade, and the water was distilled from an all-Pyrex apparatus. EXPERIMENTAL
The polystyrene latex and the protein solution were adjusted to the same pH and CKNO~, whereafter, to ensure preequilibration, they were both dialyzed for 2 days against the same aqueous solution of KNO3 of identical concentration and pH. The microcalorimeter used is a LKB 10700-2 batch type, equipped with two 18-carat gold vessels. Each vessel consists of two communicating compartments. The heat exchange between the vessels and the heat sink takes place through thermopiles that are arranged so that the difference between the heats evolved in the reaction vessel and the reference vessel is proportional to the voltage signal-time integral. Further details of the design of this microcalorimeter and of its operation have been described by Wadsb (9). Prior to each measurement the vessels were rinsed with water, ethanol, toluene, and ethanol, in that order. Then, the vessels were dried in an air stream and, just before charging, they were rinsed with dialysate. One of the vessels was charged with the latex and the protein solution in the same proportions as applied in the hydrogen ion titration experiments previously discussed (4), i.e., 4.0 cm 3 of latex (ca. 8%, w/w) in one compartment and 2.0 cm 3 of protein solution (9.0 g of HPA or 4.5 g of RNase per dm 3) in the other. The reference vessel contained 4.0 cm 3 of dialysate instead of polystyrene latex. After thermal equilibration between the vessels and the heat sink the components were mixed by rotating the vessels and the heat effect was recorded. Each experiment was preceded and succeeded by a calibration procedure, as described by Wadsb (9). To obtain the net heat of adsorption qads
Journal of Colloid and Interface Science, Vol. 66, No. 2, September1978
297
ADSORPTION OF PROTEINS V
= AadsH, the heats of dilution of both the protein solution and the polystyrene latex have to be accounted for. Since the thermopile signal reflects the difference between the heats evolved in the reaction vessel and the reference vessel, the heat of dilution of the protein is automatically subtracted. The heat of dilution of the latex was established in a separate experiment where the reaction vessel contained 4.0 cm 3 of latex and 2.0 cm 3 of dialysate and the reference vessel 4.0 cm ~ and 2.0 cm 3 of dialysate. Under all conditions studied, the dilution heat of the latex is very small. RESULTS AND DISCUSSION
Figure 1 shows a typical recording of a thermopile signal from an adsorption experiment. At fixed protein concentration the area under the curve varies proportionally with the polystyrene surface area, as expected. For all experiments the afterperiod baseline was attained within 15 min. Subsequent rotations of the calorimeter did not lead to additional heat effects, except for a very small heat of friction. This indicates that the adsorption process, at least
that part involving the enthalpy change, has completed in a fairly short period of time. The amount of protein adsorbed was determined occasionally and was found in agreement with the plateau values Fpm of the adsorption isotherms presented in Ref. (1). Enthalpy data for the adsorption of HPA and RNase are shown in the Figs. 2 and 3, respectively. AadsH is expressed per unit area of the polystyrene surface. For neither protein do the shapes of the AadsH-pH curves conform with intuitive expectation. The only part that, at least qualitatively, could be anticipated is the low pH region. Here, when the pH is decreased, the increasing electrostatic attraction between protein and latex would render the adsorption process more exothermic or less endothermic. However, at sufficiently high pH the process is again exothermic, even though under this condition the overall coulombic interaction between latex and protein is repulsive. Although qualitatively both AaosH and Fpmpass through a maximum as a function o f p H (1), suggesting a correlation between them, closer inspection shows that the positions and the trends of the two maxima as a function of o'0 do not coincide,
cn o o >
1st
rotation
2nd
rotation
time FIG. 1. Recording of a thermopile signal from the calorimeter. Journal of Colloid and Interface Science, Vol. 66, No. 2, September 1978
298
NORDE AND LYKLEMA
AadsH5
-
mJ m -2
/0 ~
/
/
/
-0~ \
",
o\
e, X i -
l"
\'k 0x
X
\\
r/> j
-'/}/ -2
.-/
\
pH
ON
x~/
•
/
Iv
-3
/
FIG. 2. Enthalpy of adsorption of HPA on polystyrene latices. ©, ~o = -15.5 /xC cm-2; A, o-o = - 6 . 3 / x C cm-2; [~, tro = - 4 . 6 p~C cm-~; V, ~o = - 2 . 3 / x C cm -~. T = 25°C. Open symbols, 0.01 M KNO3; filled symbols, 0.05 M KNO3.
so that a simple interpretation in terms of adsorbed amounts is also inadequate. Apparently, the observed trends reflect the compounding of various contributions to AadsH.
Under many conditions AadsH > 0. Realizing that the adsorption process is then still spontaneous, so that AadsG < 0, it must follow that, at least under these conditions, entropic factors constitute the driving force. Some information on the nature of a number of contributions to AadsH can be derived from closer analysis of the effects of electrolyte concentration and temperature. The influence of CKN03 on Aao~H for HPA depends in a systematic way on fro Journal of Colloid and Interface Science, Vol. 66, No. 2, September 1978
and pH. First, it is noted that the addition of KN03 always (if anything) renders the adsorption more exothermic. In the case of a latex having a small o-0, the part of the curve to the acid side of the maximum is most strongly affected by a variation of CK~o3 but for high surface charge latices the situation is reversed. If at low pH the negative value of AadsH would be due to the overall electrostatic attraction between protein and latex, addition of electrolyte would make Aad~H less negative because of screening, in contradiction with observation. It follows again that such a simple interpretation is not justified. The observed effect of electrolyte would not be in con-
299
ADSORPTION OF PROTEINS V
AadsH 5 - m J m -2
/7~ _/
°----~.~~
"-,._\
/
pH 4
" " 5
6
7
_;
8
9
"x\elO
11~,
NO
FIG. 3. E n t h a l p y o f adsorption o f R N a s e on p o l y s t y r e n e latices. (3, ~0 = - 1 5 . 5 /~C cm 2; V, ~ro = - 2 . 3 / z C c m -~. T = 25°C. O p e n s y m b o l s , 0.01 M KNO3; filled symbols, 0.05 M KNO3.
flict with our earlier finding (4) that HPA tends to adsorb with a relatively large fraction of its carboxyl groups toward the polystyrene surface. For the adsorption of RNase on the weakly charged latex the influence of CKNO~on Aad~H (pH) is insignificant, whereas for the highly charged latex it is significant though still very small. It is, therefore, probable that the variation of Fpm with C~No~ that is observed for RNase adsorbed at the high charged latex (1) results from different degrees of tilting of the adsorbed molecules rather than from different degrees of structural perturbations. The way AaasH is affected by changing the temperature, shown in the Figs. 4 and 5, is also indicative of the intricate nature of the adsorption process. Both with HPA and RNase, at first sight the dependence of A a d s r C p o n pH and tr 0 does not follow a simple pattern. However, on closer inspection some trends emerge. To that end, it is useful to review the various factors that may contribute to A a a s r C p . (i) A reduction of
hydrophobic hydration would lead to a decrease in *Cp (10, 11). (ii) The medium change of the ions transferred from the aqueous solution to the adsorbed protein layer (6), from now on referred to as the "ionic medium effect," would contribute positively to Aads0Cp, the stronger the more chaotropic the transferred ions are (11). (iii) Changes in the protein structure are a third factor, but since these are not known in detail, the resulting effect on 6Cp cannot be predicted. An increase upon adsorption of the motional freedom inside the protein molecule would lead to a positive contribution to Aads~Cp. Hence, disruption of intramolecular ion pairs, hydrogen bonds, etc., on the one hand and actual attachment of the protein molecule at the adsorbent surface on the other hand influence madsCbfpin opposite directions. In addition to these intrinsic contributions there is the rather trivial effect that the adsorbed amount Fpm itself is temperature sensitive, so that AaasH per unit area at various T's refers to different amounts of protein adJournal of Colloid and Interface Science, Vol. 66, No. 2, September 1978
300
NORDE AND LYKLEMA 5 -mJ rrr 2
S -mJm "2 A,n,H /.,
@
Co-e-Z3FC cm_2
A~H /4
(~)
0o=-155~C cm -2
,
~- /
\\
/&"
\\
,?
-3 -4
+-... ,
pH
~+\ \
_:
-5 -6
FIG. 4. Enthalpy of adsorption of HPA on polystyrene latices at different temperatures: A, 9oc; +, 25°C; ©, 37°C. CKNO3= 0.05 M.
sorbed. This is a relatively minor effect that can be taken into account because Fro(T) is known; it does not affect the sign of AadsCbCv. For the adsorption of HPA on the weakly 8
charged latex (~r0 = - 2 . 3 / ~ C cm -~) A a d s ¢ b f v derived from A a d s H a t 9 and 25°C is negative over the entire pH region investigated (see Fig. 4a). In this case, the dehydration of the hydrophobic polystyrene surface prob8 -mJ m-2 Aad~H
' m J m "2
A+dsH
o
7
o\,
6
\'x+
++ -I -2
-3 -4
-2 (~ oo:-2.31sC cm -2
-3
(~
ao=-15.5i.,tCcrn-
-4
F I G . 5. Enthalpy of adsorption of RNase on polystyrene latices at different temperatures: A, 9°C; +, 25°C; C), 37°C. CKNO~= 0.05 M.
Journal of Colloid and Interface Science, Vol. 66, No. 2, September 1978
ADSORPTION OF PROTEINS V ably dominates Aads'~Cv. At 37°C the curve for AadsH(pH) shows a different behavior between pH 4.5 and 5.8. This may be caused by a larger perturbation of the adsorbed protein structure in that pH region at 37°C, leading to a greater motional freedom inside the protein molecule. This suggestion is consistent with earlier findings concerning the temperature dependence of Fpm (1). The influence of o-0 on Aaos*Cp can be traced by comparing Figs. 4a and 4b. Adsorption on the highly charged latex (o-0 = - 1 5 . 5 /xC cm -2) results in a negative Aao~*Cp only at pH < ca. 4.7, whereas at higher pH values Aads*Ce > 0. The surface of the highly charged latex is much less hydrophobic than that of the weakly charged one. Apparently, at high pH, the sum of the contributions to AadsOCP from structural changes in the protein molecule and the ionic medium effect is positive and overcompensates the negative contribution from the dehydration of the polystyrene surface. A larger structural perturbation of the adsorbed HPA molecules (1) and a larger number of transferred ions at high pH may both contribute to the positive Aaa~*Cv. In the case of RNase adsorption, the variation of Aaa~Hwith temperature shows a pattern (Fig. 5) that is considerably different from that observed with HPA. For RNase adsorbed on the latex of low charge Aads4'Cp > 0 in the medium pH range but <0 at the extremes. In view of the discussion on the ion transfer between the solution and the adsorbed layer (6) the contribution from the ionic medium effect is expected to be maximum in the intermediate pH region. This fits in with the positive value of Aaa~*Cp over that pH range. It has been shown (1) that for RNase Fpm is only a little sensitive to the pH of adsorption. Only at extreme pH's is Fpm somewhat reduced. If these small reductions in Fpm imply less exposure of hydrophobic
301
residues of the protein molecule to the water and/or a larger extent of dehydration of the polystyrene surface due to spreading of the adsorbed molecules, this could perhaps contribute to the reversal of the sign of Aads~Cp at extreme pH. At any rate, the dominant contribution of hydrophobic dehydration, recognized in the adsorption of HPA on weakly charged latices, is not encountered here. The smaller degree of structural changes in the adsorbed RNase molecules, as compared to HPA (1), probably implies a smaller fraction of protein void volume in the adsorbed layer (7) and, hence, less dehydration of the polystyrene surface. Nonetheless, the lower hydrophobicity of the highly charged latex may help to explain the more positive value of AaasOCp for RNase adsorption at this surface. Also, the somewhat larger ionic medium effect (6) in the case of the more negative o'0 would tend to make AaasOCp more positive. Contrary to the weakly charged latex, with the highly charged one I'pm does not decrease at acid pH but it does so more markedly at alkaline pH (1). The negative value of Aaas*Cp at alkaline pH then suggests that the reduction in Fpm(pH) involves a greater extent of dehydration of hydrophobic parts of the protein molecule and/or the polystyrene surface (7). The discussion on the enthalpy data, given thus far, is only a qualitative one. At this stage the mechanism of protein adsorption is not yet understood in sufficient detail to give a reliable prediction of AadsH. H o w e v e r , A a d s H may be interpreted in a more quantitative way by assessing its constituent parts. Such an analysis will be given in Part VI (8), in which the thermodynamics of the overall adsorption process will be discussed. ACKNOWLEDGMENT
Figures 2, 3, 4, and 5 are taken from the thesis of W.N., by permission of the Editorial Board of the Communications Agricultural University Wageningen, The Netherlands. Journal of Colloid and Interface Science,
Vol. 66, No. 2, September1978
302
NORDE AND LYKLEMA REFERENCES
1. Norde, W., and Lyklema, J., J. Colloid Interface Sci. 66, 257 (1978). 2. Killmann, E., and Eckart, R., Makromol. Chemie 144, 45 (1971). 3. Nyilas, E., Chiu, T. H., and Herzlinger, G. A., Trans. Amer. Soc. Artif. Intern. Organs 20, 480 (1974). 4. Norde, W., and Lyklema, J., J. Colloid Interface Sci. 66, 266 (1978). 5. Norde, W., and Lyklema, J., J. Colloid Interface Sci. 66, 277 (1978). 6. Norde, W., and Lyklema, J., J. Colloid Interface Sci. 66, 285 (1978).
Journal of Colloid and Interface Science, Vol. 66, No. 2, September 1978
7. Norde, W., Ph.D. Thesis Agricultural University Wageningen, Netherlands, 1976; also, Commun. Agric. Univ. Wageningen, Netherlands
No. 76-6, 1976. 8. Norde, W., and Lyklema, J., Submitted for publication in J. Colloid Interface Sci. 9. Wads6, I., Acta Chem. Scand. 22, 927 (1968). 10. N6methy, G., and Scheraga, H. A., J. Chem. Phys. 36, 3401 (1962). 11. Tanford, C. "The Hydrophobic Effect." Interscience, New York, 1973. 12. Frank, H. S., and Wen, W. Y., Disc. Faraday Soc. 24, 133 (1957).
INTRODUCTION
The variation upon adsorption of the thermodynamic characteristic functions constitutes an important additional piece of information for a better understanding of the properties of proteins at interfaces. In Part I (1) of this series the irreversibility of the adsorption of human plasma albumin (HPA) and bovine pancreas ribonuclease (RNase) at polystyrene surfaces has been stressed. Because of this irreversibility, it is not valid to derive from adsorption isotherms a binding constant from which the Gibbs free energy of adsorption AadsG can be calculated. For this reason and also because in the case of protein adsorption isosteric conditions defer unambiguous definition, the physical meaning of the adsorption enthalpy calculated from the temperature dependence of the isotherms (according to Clausius-Clapeyron) is questionable. However, since the interracial
area on which, in a latex, adsorption takes place is high, the net enthalpy AadsH due to the overall adsorption process can be determined by calorimetry. For polymer adsorption only a limited number of such determinations have been reported in the literature. Killmann and Eckart (2) have established enthalpies of adsorption of polyethylene glycol on silica surfaces. Calorimetrically they found negative values, whereas the enthalpy evaluated according to the Clausius-Clapeyron equation appeared to be positive. In other words, for polymer adsorption the phenomenological isosteric heat is not a good measure for AadsH , probably because in this case it is not sufficient to characterize "isosteric" as "at constant amount adsorbed." Calorimetric determinations of AadsH for y-globulin on glass surfaces have been made by Nyilas et al. (3). For these systems AadsH per mole of protein adsorbed
295 0021-9797/78/0662-0295502.00/0 Journal of Colloid and Interface Science, Vol. 66, No. 2, September 1978
Copyright © 1978 by Academic Press,~Inc. All fights of reproduction in any form reserved.
296
NORDE AND LYKLEMA
is maximum at the surface concentration corresponding to the completion of the first layer, and it shows a sharp decay on further increasing the amount adsorbed. The authors suggest that the y-globulin molecules adsorbed in the first layer undergo the largest structural changes that could be triggered by the large adsorption enthalpy. We are inclined to interpret A a d s H in a different way. It has to be realized that AaosH is the compounded enthalpy change due to all factors, including structural alterations in the protein molecules that are involved in the overall adsorption process (1, 4-7). For some of these factors the contribution to haasH may be estimated. If the overall AadsH is experimentally obtainable, subtraction leads to the contributions to AadsH of otherwise inaccessible factors. In doing so, more information can be gained on the forces responsible for protein adsorption. This will be discussed in Part VI (8). Anticipating this treatment, we present now the results of direct microcalorimetry. In studying the adsorption mechanism it is informative to determine AadsH at various temperatures. The temperature coefficient (at constant pressure) of AadsH equals the change upon adsorption of the apparent heat capacity 6Cp of the system Aads6Cp -- { S A a d s H / S T } p .
[1]
The quantity Aads~'Cv is a measure of the structural changes that the protein molecule and the other molecules involved, i.e., water and electrolyte, undergo during the adsorption process. The experimental data presented in this paper refer to conditions of plateau-adsorption (1). The variables studied are the pH of adsorption, the surface charge tr0 of the polystyrene latex particles, the concentration CKNO,of KNO3 in solution, and the temperature T. MATERIALS
For a description of the polystyrene latices and the proteins we refer to Refs. (1)
and (7). The other chemicals were of analytical grade, and the water was distilled from an all-Pyrex apparatus. EXPERIMENTAL
The polystyrene latex and the protein solution were adjusted to the same pH and CKNO~, whereafter, to ensure preequilibration, they were both dialyzed for 2 days against the same aqueous solution of KNO3 of identical concentration and pH. The microcalorimeter used is a LKB 10700-2 batch type, equipped with two 18-carat gold vessels. Each vessel consists of two communicating compartments. The heat exchange between the vessels and the heat sink takes place through thermopiles that are arranged so that the difference between the heats evolved in the reaction vessel and the reference vessel is proportional to the voltage signal-time integral. Further details of the design of this microcalorimeter and of its operation have been described by Wadsb (9). Prior to each measurement the vessels were rinsed with water, ethanol, toluene, and ethanol, in that order. Then, the vessels were dried in an air stream and, just before charging, they were rinsed with dialysate. One of the vessels was charged with the latex and the protein solution in the same proportions as applied in the hydrogen ion titration experiments previously discussed (4), i.e., 4.0 cm 3 of latex (ca. 8%, w/w) in one compartment and 2.0 cm 3 of protein solution (9.0 g of HPA or 4.5 g of RNase per dm 3) in the other. The reference vessel contained 4.0 cm 3 of dialysate instead of polystyrene latex. After thermal equilibration between the vessels and the heat sink the components were mixed by rotating the vessels and the heat effect was recorded. Each experiment was preceded and succeeded by a calibration procedure, as described by Wadsb (9). To obtain the net heat of adsorption qads
Journal of Colloid and Interface Science, Vol. 66, No. 2, September1978
297
ADSORPTION OF PROTEINS V
= AadsH, the heats of dilution of both the protein solution and the polystyrene latex have to be accounted for. Since the thermopile signal reflects the difference between the heats evolved in the reaction vessel and the reference vessel, the heat of dilution of the protein is automatically subtracted. The heat of dilution of the latex was established in a separate experiment where the reaction vessel contained 4.0 cm 3 of latex and 2.0 cm 3 of dialysate and the reference vessel 4.0 cm ~ and 2.0 cm 3 of dialysate. Under all conditions studied, the dilution heat of the latex is very small. RESULTS AND DISCUSSION
Figure 1 shows a typical recording of a thermopile signal from an adsorption experiment. At fixed protein concentration the area under the curve varies proportionally with the polystyrene surface area, as expected. For all experiments the afterperiod baseline was attained within 15 min. Subsequent rotations of the calorimeter did not lead to additional heat effects, except for a very small heat of friction. This indicates that the adsorption process, at least
that part involving the enthalpy change, has completed in a fairly short period of time. The amount of protein adsorbed was determined occasionally and was found in agreement with the plateau values Fpm of the adsorption isotherms presented in Ref. (1). Enthalpy data for the adsorption of HPA and RNase are shown in the Figs. 2 and 3, respectively. AadsH is expressed per unit area of the polystyrene surface. For neither protein do the shapes of the AadsH-pH curves conform with intuitive expectation. The only part that, at least qualitatively, could be anticipated is the low pH region. Here, when the pH is decreased, the increasing electrostatic attraction between protein and latex would render the adsorption process more exothermic or less endothermic. However, at sufficiently high pH the process is again exothermic, even though under this condition the overall coulombic interaction between latex and protein is repulsive. Although qualitatively both AaosH and Fpmpass through a maximum as a function o f p H (1), suggesting a correlation between them, closer inspection shows that the positions and the trends of the two maxima as a function of o'0 do not coincide,
cn o o >
1st
rotation
2nd
rotation
time FIG. 1. Recording of a thermopile signal from the calorimeter. Journal of Colloid and Interface Science, Vol. 66, No. 2, September 1978
298
NORDE AND LYKLEMA
AadsH5
-
mJ m -2
/0 ~
/
/
/
-0~ \
",
o\
e, X i -
l"
\'k 0x
X
\\
r/> j
-'/}/ -2
.-/
\
pH
ON
x~/
•
/
Iv
-3
/
FIG. 2. Enthalpy of adsorption of HPA on polystyrene latices. ©, ~o = -15.5 /xC cm-2; A, o-o = - 6 . 3 / x C cm-2; [~, tro = - 4 . 6 p~C cm-~; V, ~o = - 2 . 3 / x C cm -~. T = 25°C. Open symbols, 0.01 M KNO3; filled symbols, 0.05 M KNO3.
so that a simple interpretation in terms of adsorbed amounts is also inadequate. Apparently, the observed trends reflect the compounding of various contributions to AadsH.
Under many conditions AadsH > 0. Realizing that the adsorption process is then still spontaneous, so that AadsG < 0, it must follow that, at least under these conditions, entropic factors constitute the driving force. Some information on the nature of a number of contributions to AadsH can be derived from closer analysis of the effects of electrolyte concentration and temperature. The influence of CKN03 on Aao~H for HPA depends in a systematic way on fro Journal of Colloid and Interface Science, Vol. 66, No. 2, September 1978
and pH. First, it is noted that the addition of KN03 always (if anything) renders the adsorption more exothermic. In the case of a latex having a small o-0, the part of the curve to the acid side of the maximum is most strongly affected by a variation of CK~o3 but for high surface charge latices the situation is reversed. If at low pH the negative value of AadsH would be due to the overall electrostatic attraction between protein and latex, addition of electrolyte would make Aad~H less negative because of screening, in contradiction with observation. It follows again that such a simple interpretation is not justified. The observed effect of electrolyte would not be in con-
299
ADSORPTION OF PROTEINS V
AadsH 5 - m J m -2
/7~ _/
°----~.~~
"-,._\
/
pH 4
" " 5
6
7
_;
8
9
"x\elO
11~,
NO
FIG. 3. E n t h a l p y o f adsorption o f R N a s e on p o l y s t y r e n e latices. (3, ~0 = - 1 5 . 5 /~C cm 2; V, ~ro = - 2 . 3 / z C c m -~. T = 25°C. O p e n s y m b o l s , 0.01 M KNO3; filled symbols, 0.05 M KNO3.
flict with our earlier finding (4) that HPA tends to adsorb with a relatively large fraction of its carboxyl groups toward the polystyrene surface. For the adsorption of RNase on the weakly charged latex the influence of CKNO~on Aad~H (pH) is insignificant, whereas for the highly charged latex it is significant though still very small. It is, therefore, probable that the variation of Fpm with C~No~ that is observed for RNase adsorbed at the high charged latex (1) results from different degrees of tilting of the adsorbed molecules rather than from different degrees of structural perturbations. The way AaasH is affected by changing the temperature, shown in the Figs. 4 and 5, is also indicative of the intricate nature of the adsorption process. Both with HPA and RNase, at first sight the dependence of A a d s r C p o n pH and tr 0 does not follow a simple pattern. However, on closer inspection some trends emerge. To that end, it is useful to review the various factors that may contribute to A a a s r C p . (i) A reduction of
hydrophobic hydration would lead to a decrease in *Cp (10, 11). (ii) The medium change of the ions transferred from the aqueous solution to the adsorbed protein layer (6), from now on referred to as the "ionic medium effect," would contribute positively to Aads0Cp, the stronger the more chaotropic the transferred ions are (11). (iii) Changes in the protein structure are a third factor, but since these are not known in detail, the resulting effect on 6Cp cannot be predicted. An increase upon adsorption of the motional freedom inside the protein molecule would lead to a positive contribution to Aads~Cp. Hence, disruption of intramolecular ion pairs, hydrogen bonds, etc., on the one hand and actual attachment of the protein molecule at the adsorbent surface on the other hand influence madsCbfpin opposite directions. In addition to these intrinsic contributions there is the rather trivial effect that the adsorbed amount Fpm itself is temperature sensitive, so that AaasH per unit area at various T's refers to different amounts of protein adJournal of Colloid and Interface Science, Vol. 66, No. 2, September 1978
300
NORDE AND LYKLEMA 5 -mJ rrr 2
S -mJm "2 A,n,H /.,
@
Co-e-Z3FC cm_2
A~H /4
(~)
0o=-155~C cm -2
,
~- /
\\
/&"
\\
,?
-3 -4
+-... ,
pH
~+\ \
_:
-5 -6
FIG. 4. Enthalpy of adsorption of HPA on polystyrene latices at different temperatures: A, 9oc; +, 25°C; ©, 37°C. CKNO3= 0.05 M.
sorbed. This is a relatively minor effect that can be taken into account because Fro(T) is known; it does not affect the sign of AadsCbCv. For the adsorption of HPA on the weakly 8
charged latex (~r0 = - 2 . 3 / ~ C cm -~) A a d s ¢ b f v derived from A a d s H a t 9 and 25°C is negative over the entire pH region investigated (see Fig. 4a). In this case, the dehydration of the hydrophobic polystyrene surface prob8 -mJ m-2 Aad~H
' m J m "2
A+dsH
o
7
o\,
6
\'x+
++ -I -2
-3 -4
-2 (~ oo:-2.31sC cm -2
-3
(~
ao=-15.5i.,tCcrn-
-4
F I G . 5. Enthalpy of adsorption of RNase on polystyrene latices at different temperatures: A, 9°C; +, 25°C; C), 37°C. CKNO~= 0.05 M.
Journal of Colloid and Interface Science, Vol. 66, No. 2, September 1978
ADSORPTION OF PROTEINS V ably dominates Aads'~Cv. At 37°C the curve for AadsH(pH) shows a different behavior between pH 4.5 and 5.8. This may be caused by a larger perturbation of the adsorbed protein structure in that pH region at 37°C, leading to a greater motional freedom inside the protein molecule. This suggestion is consistent with earlier findings concerning the temperature dependence of Fpm (1). The influence of o-0 on Aaos*Cp can be traced by comparing Figs. 4a and 4b. Adsorption on the highly charged latex (o-0 = - 1 5 . 5 /xC cm -2) results in a negative Aao~*Cp only at pH < ca. 4.7, whereas at higher pH values Aads*Ce > 0. The surface of the highly charged latex is much less hydrophobic than that of the weakly charged one. Apparently, at high pH, the sum of the contributions to AadsOCP from structural changes in the protein molecule and the ionic medium effect is positive and overcompensates the negative contribution from the dehydration of the polystyrene surface. A larger structural perturbation of the adsorbed HPA molecules (1) and a larger number of transferred ions at high pH may both contribute to the positive Aaa~*Cv. In the case of RNase adsorption, the variation of Aaa~Hwith temperature shows a pattern (Fig. 5) that is considerably different from that observed with HPA. For RNase adsorbed on the latex of low charge Aads4'Cp > 0 in the medium pH range but <0 at the extremes. In view of the discussion on the ion transfer between the solution and the adsorbed layer (6) the contribution from the ionic medium effect is expected to be maximum in the intermediate pH region. This fits in with the positive value of Aaa~*Cp over that pH range. It has been shown (1) that for RNase Fpm is only a little sensitive to the pH of adsorption. Only at extreme pH's is Fpm somewhat reduced. If these small reductions in Fpm imply less exposure of hydrophobic
301
residues of the protein molecule to the water and/or a larger extent of dehydration of the polystyrene surface due to spreading of the adsorbed molecules, this could perhaps contribute to the reversal of the sign of Aads~Cp at extreme pH. At any rate, the dominant contribution of hydrophobic dehydration, recognized in the adsorption of HPA on weakly charged latices, is not encountered here. The smaller degree of structural changes in the adsorbed RNase molecules, as compared to HPA (1), probably implies a smaller fraction of protein void volume in the adsorbed layer (7) and, hence, less dehydration of the polystyrene surface. Nonetheless, the lower hydrophobicity of the highly charged latex may help to explain the more positive value of AaasOCp for RNase adsorption at this surface. Also, the somewhat larger ionic medium effect (6) in the case of the more negative o'0 would tend to make AaasOCp more positive. Contrary to the weakly charged latex, with the highly charged one I'pm does not decrease at acid pH but it does so more markedly at alkaline pH (1). The negative value of Aaas*Cp at alkaline pH then suggests that the reduction in Fpm(pH) involves a greater extent of dehydration of hydrophobic parts of the protein molecule and/or the polystyrene surface (7). The discussion on the enthalpy data, given thus far, is only a qualitative one. At this stage the mechanism of protein adsorption is not yet understood in sufficient detail to give a reliable prediction of AadsH. H o w e v e r , A a d s H may be interpreted in a more quantitative way by assessing its constituent parts. Such an analysis will be given in Part VI (8), in which the thermodynamics of the overall adsorption process will be discussed. ACKNOWLEDGMENT
Figures 2, 3, 4, and 5 are taken from the thesis of W.N., by permission of the Editorial Board of the Communications Agricultural University Wageningen, The Netherlands. Journal of Colloid and Interface Science,
Vol. 66, No. 2, September1978
302
NORDE AND LYKLEMA REFERENCES
1. Norde, W., and Lyklema, J., J. Colloid Interface Sci. 66, 257 (1978). 2. Killmann, E., and Eckart, R., Makromol. Chemie 144, 45 (1971). 3. Nyilas, E., Chiu, T. H., and Herzlinger, G. A., Trans. Amer. Soc. Artif. Intern. Organs 20, 480 (1974). 4. Norde, W., and Lyklema, J., J. Colloid Interface Sci. 66, 266 (1978). 5. Norde, W., and Lyklema, J., J. Colloid Interface Sci. 66, 277 (1978). 6. Norde, W., and Lyklema, J., J. Colloid Interface Sci. 66, 285 (1978).
Journal of Colloid and Interface Science, Vol. 66, No. 2, September 1978
7. Norde, W., Ph.D. Thesis Agricultural University Wageningen, Netherlands, 1976; also, Commun. Agric. Univ. Wageningen, Netherlands
No. 76-6, 1976. 8. Norde, W., and Lyklema, J., Submitted for publication in J. Colloid Interface Sci. 9. Wads6, I., Acta Chem. Scand. 22, 927 (1968). 10. N6methy, G., and Scheraga, H. A., J. Chem. Phys. 36, 3401 (1962). 11. Tanford, C. "The Hydrophobic Effect." Interscience, New York, 1973. 12. Frank, H. S., and Wen, W. Y., Disc. Faraday Soc. 24, 133 (1957).