Electrochemical Studies of the Effect of Temperature and pH on the

Department of Chemistry, Acadia University, Wolfville, Nova Scotia B0P 1X0, Canada. Received March 13, 2000. In Final Form: July 7, 2000. The adsorpti...
1 downloads 0 Views 102KB Size
Download PDF
8480

Langmuir 2000, 16, 8480-8488

Electrochemical Studies of the Effect of Temperature and pH on the Adsorption of r-Lactalbumin at Pt Nora R. Cabilio, Sasha Omanovic, and Sharon G. Roscoe* Department of Chemistry, Acadia University, Wolfville, Nova Scotia B0P 1X0, Canada Received March 13, 2000. In Final Form: July 7, 2000 The adsorption behavior of holo- and apo-R-lactalbumin at a Pt/electrolyte interface was studied in an acidic, neutral, and alkaline medium over the temperature range 273 to 353 K, using the cyclic voltammetry technique. It was shown that the surface charge density, resulting from protein adsorption, is directly proportional to the amount of adsorbed protein (surface concentration), indicating that adsorption is accompanied by the transfer of charge. A significant difference in the amount of adsorbed protein (between the two types of protein) was obtained at pH 7 because of the difference in conformation of the molecule in the presence/absence of bound calcium. On the other hand, the resemblance in behavior of the two types of proteins observed at pH 2 and pH 11 suggested that the protein is in its molten globule state and is depleted in calcium at these pHs. The adsorption process was modeled using the Langmuir adsorption isotherm. The values of the Gibbs free energy of adsorption indicated that the protein molecules strongly adsorb onto the Pt surface via chemisorption. The protein expressed the highest affinity toward adsorption at pH 2 and the lowest at pH 11. The adsorption process was found to be endothermic, resulting from the excess energetics required for the breaking of intramolecular interactions relative to those involved in the formation of protein-metal bonds. The adsorption of R-LA onto a Pt surface was found to be an entropically governed process, suggesting structural unfolding of the protein at the electrode surface.

Introduction Most proteins are highly surface active; in other words, they have a strong tendency to adsorb at a solid/liquid interface. This phenomenon is of great relevance in a wide variety of both technical and natural systems. Although the adsorption of proteins has been extensively studied in recent years, the process is not yet completely understood because of the intrinsic complexity of the process itself. The heating of biological fluids in the food industry often causes deposition (fouling) on the processing equipment. A deposit may consist of salts, proteins, and various other organic molecules. However, it is generally agreed that the adsorption of proteins plays one of the most important roles in the overall process of fouling of the metallic surfaces because of their heat sensitivity and high concentration in foodstuff. For cases such as these, it is desirable to minimize protein adsorption. The dairy industry has been confronted with the fouling of metal surfaces since plate heat exchangers were introduced in the process of pasteurization and sterilization of milk.1,2 A large number of investigations to better understand the process of fouling have been reported,3-5 but a real breakthrough in the complete control of fouling has not been reached. This is mainly because of the complexity of the dairy systems and a lack of understanding of the mechanism of fouling. However, it is generally agreed that the adsorption of whey proteins plays one of the most important roles in the overall process of fouling of the metallic surfaces. Therefore, the more that is learned about the adsorption of whey proteins onto metal surfaces and the conformational changes of the proteins which affect adsorption, the more that can be learned about the possible (1) Dejong, P. Trends Food Sci. Technol. 1997, 8, 401. (2) Changani, S. D.; Belmarbeiny, M. T.; Fryer, P. J. Exp. Thermal and Fluid Sci. 1997, 14, 392. (3) Addesso, A.; Lund, D. B. J. Food Process. Preserv. 1997, 21, 319. (4) Visser, H. J. Dispersion Sci. Technol. 1998, 19, 1127. (5) Kim, J. C.; Lund, D. B. J. Food Process Eng. 1998, 21, 369.

removal of these proteins to prevent fouling of the process equipment. One of the major whey proteins is R-lactalbumin (R-LA),6 and it is also one of the main constituents of milk deposits on heat exchangers. Although many other substances are involved in milk deposit formation, knowledge of the adsorption behavior of R-LA onto solid surfaces would greatly contribute to the understanding of the deposit formation mechanism. The R-lactalbumin (R-LA) molecule consists of a single polypeptide chain (Mr ) 142007) and four disulfide bonds.8 It is a compact globular protein that binds the calcium ion in a 1:1 ratio at a specific binding site and consists of two domains: an R-helical domain and a β-sheet domain.7 The metal ion binds to the protein via seven oxygen atoms to form a distorted pentagonal bipyramid.9 These oxygen atoms originate from two main chain carbonyl groups (Lys79 and Asp-84), three side chain carboxyl groups (Asp-82, 87, 88), and two water molecules.9 R-LA is present in mammalian milk and functions as a specificity modifier of an enzyme, galactosyltransferase.10,11 It is genetically and structurally homolegous to c-type lysozyme.12 The structural and functional properties of R-LA and its interrelationships with lysozyme are excellently reviewed by McKenzie and White11 and by Kronman.13 A remarkable property of R-LA as a model of protein folding studies is the high stability of its molten globule (MG) state,14 which is a conformation generally defined (6) Visser, J.; Jeurnink, T. J. M. Exp. Thermal and Fluid Sci. 1997, 14, 407. (7) Kuwajima, K. FASEB J. 1996, 10, 102. (8) Morr, C. V.; Ha, E. Y. W. Crit. Rev. Food Sci. Nutr. 1993, 33, 431. (9) Mizuguchi, M.; Nara, M.; Kawano, K.; Nitta, K. FEBS Lett. 1997, 417, 153. (10) Hiroaka, Y.; Segawa, T.; Kuwajima, K.; Sugai, S.; Murai, N. Biochem. Biophys. Res. Commun. 1980, 95, 1098. (11) McKenzie, H. A.; White, F. H., Jr. Adv. Protein Chem. 1991, 41, 173. (12) Hill, R. L.; Brew, K. Adv. Enzymol. Relat. Areas Mol. Biol. 1975, 43, 411. (13) Kronman, M. J. CRC Crit. Rev. Biochem. Mol. Biol. 1989, 24, 565. (14) Kuwajima, K. Proteins 1989, 6, 87.

10.1021/la0003948 CCC: $19.00 © 2000 American Chemical Society Published on Web 09/28/2000

Adsorption of R-Lactalbumin at Pt+

as having a native-like secondary structure and a more relaxed or disordered tertiary structure.14,15 The molten globule state of R-LA has been studied extensively. It is a highly heterogeneous state characterized as having a largely ordered alpha helical domain and a more disordered beta sheet domain than the native (N) protein.7 The hydrophobic core of the protein is more hydrated and becomes more exposed in the molten globule state which may be useful in allowing the protein to pass through membranes in the body.16,17 Because of the relaxed side chains, the MG state of R-LA is ∼10-20% larger in volume7 than that of the native protein. This MG state has been known to occur in conditions of extreme pH, at increased temperatures, in the presence of mild denaturants, and upon the removal of the calcium ion that is associated with the molecule.7,13 The states that occur under each of these conditions may not be exactly similar because of the highly heterogeneous nature of the MG state. It has been postulated that the destabilization of the calcium-rich protein (holo-R-LA) upon release of the ion is the result of the unfavorable electrostatic interactions of charged groups in the calcium-binding loop.18 Recently, we have investigated the adsorption behavior of some globular proteins, such as β-lactoglobulin,19-21 Cadepleted R-lactalbumin,22 κ-casein,23 ribonuclease,20,21 lysozyme,20,21 BSA,22 insulin,24 cytochrome c,25 myoglobin,25 and hemoglobin25 at a platinum surface and BSA and β-lactoglobulin at a stainless steel surface.26 The adsorption behavior of BSA and fibrinogen, which is a fibrous protein, has been investigated at a commercially pure titanium surface.27 It was found that the interactions between proteins and surfaces, resulting in adsorption, can be affected by a number of factors, such as temperature, conformation of the protein in solution and its bulk concentration, pH, ionic strength, and the surface characteristics of the material onto which adsorption occurs. In the present paper, we report on the adsorption behavior of Ca-rich R-LA (holo-type) and Ca-depleted R-LA (apo-type) onto a platinum electrode in a wide temperature range, from 273 to 353 K, and in an acidic (pH 2), neutral (pH 7), and alkaline (pH 11) medium. The adsorption of the protein to the surface, directly from an aqueous solution, is discussed on the basis of cyclic voltammetry measurements. Saturated surface concentrations and thermodynamic adsorption values are presented and discussed. Experimental Section Reagents and Solutions. The stock solutions of bovine R-lactalbumin (Sigma Chemical Company, product no. L-6010, type III, calcium depleted, and product no. L-5385, type I) were (15) Ptitsyn, O. B. In Protein Folding; Creighton, T. E., Ed., Freeman, New York, 1992; pp 243-300. (16) Lala, A. K.; Kaul, P.; Bharata Ratnam, B. J. Protein Chem. 1995, 14, 601. (17) Ban˜uelos, S.; Muga, A. Biochemistry 1996, 35, 3892. (18) Griko, Y. V.; Remeta, D. P. Protein Sci. 1999, 8, 554. (19) Roscoe, S. G.; Fuller, K. L.; Robitaille, G. J. Colloid Interface Sci. 1993, 160, 243. (20) Roscoe, S. G.; Fuller, K. L. J. Colloid Interface Sci. 1992, 152, 429. (21) Roscoe, S. G. In Modern Aspects of Electrochemistry; Bockris, J. O’M., Conway, B. E., White, R. E., Eds.; Plenum Press: New York, 1996; Vol. 29, p 319. (22) Rouhana, R.; Budge, S. M.; MacDonald, S. M.; Roscoe, S. G. Food Res. Int. 1997, 30, 303. (23) Roscoe, S. G.; Fuller, K. L. Food Res. Int. 1993, 26, 343. (24) MacDonald, S. M.; Roscoe, S. G. J. Colloid Interface Sci. 1996, 184, 449. (25) Hanrahan, K. L.; MacDonald, S. M.; Roscoe, S. G. Electrochim. Acta 1996, 41, 2469. (26) Omanovic, S.; Roscoe, S. G. Langmuir 1999, 15, 8315. (27) Jackson, D. R.; Omanovic, S.; Roscoe, S. G. Langmuir 2000, 16, 5449.

Langmuir, Vol. 16, No. 22, 2000 8481 prepared by dissolving a massed amount of solid R-LA in either 0.05 M phosphate buffer solution (pH 7.0 or pH 10.9) or 0.05 M sulfuric acid solution (near pH 2). Although the sulfuric acid solution was not a buffer, the pH level remained below pH 3.8 during the experiment, which is the acidity needed to ensure that the protein is in its molten globule state. The phosphate buffer was previously prepared using anhydrous potassium phosphate (KH2PO4, cell culture tested for pH 7.0; K2HPO4, cell culture tested for pH 10.9) and sodium hydroxide (prepared from standardized BDH Chemical Company concentrate), while the sulfuric acid solution was made from standardized Baker analyzed ultrapure concentrated sulfuric acid. Conductivity water (Nanopure, resistivity 18.2 MΩ cm) was used in the preparation of all aqueous solutions. Working solutions of various R-LA concentrations were prepared by mixing the required amount of the stock solution and phosphate buffer or sulfuric acid solution. Electrochemical Equipment. A three-compartment glass cell was constructed with two glass-sleeved stopcocks to separate the compartments. The working and counter electrodes were composed of high purity platinum wire (99.99%, JohnsonMatthey) and were degreased by refluxing in acetone, sealed in soft glass, electrochemically cleaned by potential cycling in 0.5 M sulfuric acid, and stored in 98% sulfuric acid. The real surface area of the working electrode was obtained from the charge under the hydrogen underpotential deposition peaks when in 0.5 M sulfuric acid.28 A saturated calomel reference electrode (SCE) was constructed by a standard procedure.29 All potentials in this paper are referred to the SCE. Cyclic voltammograms at pH 2 and 7 were obtained by using a Hokuto Denko potentiostat (model HA-301) and a Hokuto Denko function generator (model HB-111) and recorded on an Allen Datagraph Inc. X-Y recorder-plotter (model 720M). Cyclic voltammograms at pH 11 were obtained by using an EG&G PAR VersaStat potentiostat/galvanostat controlled by a PC. The EG&G PAR model 270/250 research electrochemistry software, version 4.3, was used for cyclic voltammetry measurements. The sweep rate used throughout was 500 mV s-1. Experimental Methodology. All measurements were carried out in an oxygen-free solution, which was achieved by continuous purging of the cell with argon gas (Praxair). This bubbling also provided a well-mixed bulk solution. The protein solution was prepared in a separate container using the required buffer or solution for the specific experiment and was allowed to equilibrate for at least 30 min in the constant-temperature bath, fitted with a Julabo P temperature regulator, at the same temperature as the electrochemical cell. After the electrode was characterized for the electrochemical technique in the phosphate buffer or sulfuric acid solution, aliquots of protein were then added to the electrochemical cell and the electrochemical measurements were repeated with each aliquot. The surface charge density, Qads, resulting from the deposition of the protein on the electrode after additions of aliquots into the bulk solution, was determined as described previously.19-25 The surface charge density, Qads, was determined from the difference between the areas of anodic oxidation and reduction in the presence of the adsorbing protein, after subtracting the small difference between these two areas in the absence of protein, as follows:

Qads ) [QOoP - QOrP] - [QOo - QOr]

(1)

where QOo (C cm-2) is the anodic oxidation charge density in phosphate buffer or sulfuric acid solution, QOr is the oxide reduction charge density in phosphate buffer or sulfuric acid solution, QPOo is the anodic oxidation charge density, andQPOr is the oxide reduction charge density in the presence of the protein. Cyclic voltammograms were recorded after each aliquot of concentrated protein solution was added to the electrolyte and allowed to equilibrate to a steady-state condition. (28) Angerstein-Kozlowska, H. In Comprehensive Treatise of Electrochemistry; Bockris, J. O’M., Conway, B. E., Sarangapani, S., Eds.; Plenum Press: New York, 1984; Vol. 9, pp 15-59. (29) Kennedy, J. H. In Analytical Chemistry; Harcourt Brace Jovanovich, San Diego, CA, 1984; pp 482-484.

8482

Langmuir, Vol. 16, No. 22, 2000

Cabilio et al.

Figure 1. Surface charge density as a function of concentration of holo-R-LA in phosphate buffer, pH 7: (b) 273; (O) 299; (1) 313; (3) 323; (9) 333; (0) 343; and (() 353 K.

Results and Discussion Measurements at pH 7. Figure 1 shows the dependence of the surface charge density, Qads, resulting from the adsorption of holo-R-LA (calcium-rich protein) onto a Pt surface, on the equilibrium concentration of the protein in the bulk solution at pH 7 and different temperatures. Using eq 1, Qads was calculated from the voltammograms recorded with an anodic potential limit of +0.50 V, which, in a protein-free solution, resulted in a monolayer surface coverage with OH species.19-22,24 This allows us to compare our results with those obtained by other researchers using nonelectrochemical techniques. It can be seen from Figure 1 that the adsorption behavior of holo-R-LA is similar to that of apo-R-LA (calcium-depleted protein) shown in Figure 2 in ref 22. In both cases, as the concentration of the protein was increased in the bulk solution, the surface charge density rapidly increased and reached a plateau level at ∼0.015 g L-1 at all the temperatures. In Figure 1, the solid lines do not represent any attempt to model the data but are shown to aid in visual presentation. Using surface charge density values, Qads, it is possible to calculate the protein surface concentration, Γ,19-25

Γ)

QadsMr nF

(2)

where Qads is the surface charge density in C cm-2, Mr is the molar mass of the protein in g mol-1, n is the number of electrons transferred, and F is the Faraday constant in C mol-1. Experiments performed in our laboratory, using a Pt electrode,19-25 indicated a direct involvement of carboxylate groups of proteins in the adsorption processes at the anodic Pt surface. Further, on the basis of the infrared reflection-absorption spectroscopy and ellipsometry experiments, Liedberg et al.30 concluded that the binding of the carboxyl groups of β-lactoglobulin to a metal is through a chemical linkage (formation of an ester-type bond). Horanyi et al.31 concluded that when a carboxyl group is present, the adsorption properties of a molecule are entirely determined by this group. Similar conclusions of protein interaction with surfaces through the carboxylate groups are reported in the literature.19-25,32-37 There(30) Liedberg, B.; Invarsson, B.; Hegg, P. O.; Lundstro¨m, I. J. Colloid Interface Sci. 1986, 114, 386. (31) Horanyi, G.; Vertes, G.; Rizmayer, E. M. J. Electroanal. Chem. 1973, 48, 207. (32) Hansen, D. C.; Luther, G. W., III; Waite, J. H. J. Colloid Interface Sci. 1994, 168, 206.

fore, in eq 2, the number of electrons transferred refers to the number of carboxylate groups in R-LA available for binding to the Pt surface, and it has been shown19-27 that the overall reaction involves a two-electron-transfer process per carboxylate group. The number of carboxylate groups in R-LA is 21 (20 carboxylate groups from acidic amino acid residues and one from the carboxylate terminus of the protein).38,39 However, Ca2+ ion is bound to four carboxyl groups (Asp-82, 84, 87, 88),9 and therefore, the total number of carboxylate groups available for binding of holo-R-LA to the Pt surface is 17. Using the above data and eq 2, the saturated surface concentration (from the plateau) for holo-R-LA on Pt at 299 K (Figure 1) was calculated to be 2.9 ( 0.1 mg m-2, which is a considerably larger value than the concentration calculated for apoR-LA, 1.7 ( 0.3 mg m-2.22 The origin of the observed difference comes partially from the fact that apo-R-LA, which is in a molten globule state due to the lack of Ca2+ ions, occupies about a 10-20% larger volume than that of holo-R-LA7 and, consequently, its adsorption results in a lower value of the saturated surface concentration. On the other hand, saturated surface concentration values reported in the literature significantly vary, depending on physical and chemical properties of an absorbent surface, temperature, pH, ionic strength of a solution, etc. From an isotherm for adsorption of apo-R-LA onto a hydrophobic silicon surface reported in Suttiprasit et al.,40 a saturated surface concentration value obtained at 300 K and pH 7 was calculated to be ∼3.3 mg m-2, and a similar value (3 mg m-2) was also reported by Matsumura et al.41 for the adsorption at an oil-water interface. Galisteo et al.42 investigated the adsorption of apo-R-LA onto an AgI surface and reported a maximum saturated surface concentration value ∼1.7 mg m-2. Addesso et al.3 have concluded that there is no significant difference between saturated surface concentration values when apoR-LA adsorbs on hydrophilic (stainless steels 304/316 and titanium) or hydrophobic (Teflon) surfaces at pH 7. The results obtained on hydrophilic and hydrophobic silicon surfaces by Krisdhasima et al.43 point to the same conclusion. However, it is important to note that the apo-type of R-LA used in this paper did not actually represent a true apo-form of the protein, since it was partially stabilized by the presence of Na+ and K+ ions in the working electrolyte that are also known to specifically bind to the R-LA molecule.18 Even at low concentrations, sodium may generate a broad spectrum of native as well as intermediate denatured conformations, which is especially relevant for the structural and thermodynamic characterization of the molten globule state of R-LA. The results presented in Figure 1 show that, with an increase in temperature, the plateau surface concentration (33) Fukuzaki, S.; Urano, H.; Nagata, K. J. Ferment. Bioeng. 1995, 80, 6; 1996, 81, 163. (34) Marangoni, D. G.; Smith, R. S.; Roscoe, S. G. Can. J. Chem. 1989, 67, 921. (35) Marangoni, D. G.; Wylie, I. G. N.; Roscoe, S. G. Bioelectrochem. Bioenerg. 1991, 25, 269. (36) Wylie, I. G. N.; Roscoe, S. G. Bioelectrochem. Bioenerg. 1992, 28, 367. (37) MacDonald, S. M.; Roscoe, S. G. Electrochim. Acta 1997, 42, 1189. (38) Pike, A. C. W.; Brew, K.; Acharya, K. R. Structure 1996, 4, 691. (39) Calderone, V.; Giuffrida, M. G.; Viterbo, D.; Napolitano, L.; Fortunato, D.; Conti, A.; Acharya, K. R. FEBS Lett. 1996, 394, 91. (40) Suttiprasit, P.; Krisdhasima, V.; McGuire, J. J. Colloid Interface Sci. 1992, 154, 316. (41) Matsumura, Y.; Mitsui, S.; Dickinson, E.; Mori, T. Food Hydrocolloids 1994, 8, 555. (42) Galisteo, F.; Norde, W. J. Colloid Interface Sci. 1995, 172, 502. (43) Krisdhasima, V.; Vinaraphong, P.; McGuire J. J. Colloid Interface Sci. 1993, 161, 325.

Adsorption of R-Lactalbumin at Pt+

Langmuir, Vol. 16, No. 22, 2000 8483

Figure 2. Plateau surface concentration of (O) holo- and (4) apo-R-LA at various temperatures obtained in phosphate buffer, pH 7. Inset shows a percentile dependence of the surface Pt oxide blocked by the adsorption of the protein.

increases, too. This is clearly shown in Figure 2, which represents the dependence of the plateau surface concentration values of both types of R-LA on the temperature. The inset in the same figure illustrates the blocking effect of protein layers toward oxide-monolayer formation, which was calculated from oxide-reduction charge values44

(

% oxide blocked ) 1 -

)

QOrP × 100% QOr

(3)

where QPOr is the oxide-reduction charge recorded at the saturation (plateau) surface concentration of the protein and QOr is the reduction charge recorded in the proteinfree phosphate buffer solution. The curves in Figure 2 demonstrate that, with an increase in temperature, the saturated surface concentration, Γmax, of both types of protein gradually increases. At the same time, the blocking effect of the protein layers toward oxide-monolayer formation also increases in a similar manner (see the inset). It is important to note that, in the whole temperature range, the Γmax values of holo-R-LA are larger in comparison with the values obtained with apo-R-LA. Both curves are almost parallel in the temperature range from 273 to 333 K. Above this value, the saturated surface concentration of the holo-type continues to increase, while the value for the apo-type remains almost constant. The inset in Figure 2 shows that, with an increase in temperature, the blocking effect of both forms of the protein increases. The two curves are again parallel in the temperature range from 273 K to 333 K. At the two lowest temperatures, the blocking effect of both types of proteins is low, but above 299 K, it abruptly increases. This increase could be related to the formation of a multilayer structure of the adsorbed protein at the surface. According to the dimensions of the protein molecule (4.3 × 3.2 × 2.8 nm3)40,42 it appears that, at 273 and 299 K, the Pt surface is covered with a monolayer of adsorbed protein molecules, since a value calculated using the above dimensions is 1.7 mg m-2. However, the increase in temperature facilitates further adsorption of both types of proteins and formation of the second protein layer above 299 K. The inset shows that above 333 K the blocking effect of the protein layers abruptly diminishes (see the peaks at 343 K), while, at the same time, the surface concentration of the apo-type (44) Petrie, R. J.; MacDonald, S. M.; Fuller, K. L.; Roscoe, S. G. Can. J. Chem. 1997, 75, 1585.

remains relatively constant and that of the holo-type further increases. This indicates that some structural and conformational changes occur above 333 K and coincide with the potential region of the thermal denaturation of the protein.45 Above 343 K, the blocking effect abruptly increases, which could be a result of rearrangement of the molecules at the surface after the denaturation is completed. For the apo-type, the surface concentration still remains unchanged, thus indicating that there is no further adsorption of the protein onto the Pt-surface (detectable by the charge-transfer measurements). However, this does not exclude further noncharge-exchange related adsorption (i.e., physiosorption) of the protein on the preexisting adsorption layer. Therefore, the increase in the blocking effect of the adsorbed protein layer (apotype) at 353 K unaccompanied with the corresponding increase in the saturated surface concentration observed in Figure 2 indicates possible formation of the third adsorption layer via physiosorption. On the other hand, the surface concentration of the holo-type sharply increases at 353 K, which indicates further adsorption of this type of protein via exchange of charge with the Pt surface. The curves in Figure 2 suggest that there is no release of Ca2+ upon adsorption and/or denaturation of holo-RLA at the Pt surface, since the curves do not overlap even at high temperatures. Even if we assume that above 333 K holo-R-LA releases calcium and if we then take additional four carboxylate groups into account (which were bound to the calcium ion) when calculating the surface concentration using eq 2, the obtained surface concentration value for the holo-type is still higher than that one for the apo-type (see dashed line in Figure 2 above 333 K). This conclusion is supported by the results reported by Vanderheeren et al.,46 who found, using circular dichroism measurements, that Ca2+ remains associated with R-LA after thermal unfolding of its tertiary structure. Such conclusion was also drawn by Kuroki et al.47 from a calorimetric study on a mutant human lysozyme containing the R-LA Ca2+ binding site. Haynes et al.48 have shown that adsorption of R-LA at polystyrenewater interfaces results in a significant change in its secondary and tertiary structure, which does not correspond to the transition to a molten globule state which is promoted by Ca2+ release. Among the several isotherms tested, the Langmuir adsorption isotherm was shown to best describe adsorption of the proteins onto solid surfaces, which is in agreement with the literature.32,33,49-51 Its mathematical description is

Γ)

BadsΓmaxc 1 + Badsc

(4)

in which c (mol cm-3) is the equilibrium concentration of the adsorbate in the bulk solution, Γ (mol cm-2) is the amount of protein adsorbed (i.e., surface concentration), (45) Arnebrant, T.; Barton, K.; Nylander, T. J. Colloid Interface Sci. 1987, 119, 383. (46) Vanderheeren, G.; Ganssens, I. J. Biol. Chem. 1994, 269, 7090. (47) Kuroki, R.; Kawakita, S.; Nakamura, H.; Yutani, K. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 6803. (48) Haynes, C. A.; Sliwinsky, E.; Norde, W. J. Colloid Interface Sci. 1994, 164, 394. (49) Brash, J. L.; ten Hove, P. J. Biomater. Sci. Polym. Ed. 1993, 4, 591. (50) Klinger, A.; Steinberg, D.; Kohavi, D.; Sela, M. N. J. Biomed. Mater. Res. 1997, 36, 387. (51) LeDuc, C.; Ten Hove, P.; Park, S.; Vroman, L.; Brash, J.; Leonard, E. F. J. Biomater. Sci. Polymer Ed. 1995, 7, 531.

8484

Langmuir, Vol. 16, No. 22, 2000

Cabilio et al.

Figure 3. Adsorption isotherm of (O) holo- and (4) apo-R-LA adsorbed onto Pt in phosphate buffer, pH 7 at 313 K. Symbols are experimental values, and solid lines are the best fit. Inset: experimental data (symbols) fitted using the Langmuir isotherm equation and calculated parameters (Γmax and Bads).

Γmax (mol cm-2) is the maximum value of Γ (plateau or saturated surface concentration), and the parameter Bads (cm3 mol-1) reflects the affinity of the adsorbate molecules toward adsorption sites. Equation 4 can be rearranged to give

1 c c ) + Γ BadsΓmax Γmax

(5)

If the Langmuir isotherm is valid for an observed system, a plot of c/Γ versus concentration c should yield a straight line with parameters Γmax and Bads derived from the slope and intercept, respectively. As an example, adsorption of both forms of R-LA is presented in Figure 3, and indeed, the c/Γ versus c dependence is linear, with a correlation coefficient r2 ) 0.9988 for holo-R-LA and r2 ) 0.9972 for apo-R-LA. The calculated maximum surface concentration, Γmax, was found to be 4.0 ( 0.4 mg m-2 for holo-R-LA, which is close to the experimentally obtained value of 3.95 mg m-2 (Figure 2). The value of the maximum surface concentration for apo-R-LA was calculated to be 2.3 ( 0.3 mg m-2, which is also quite comparable to the experimentally obtained value of 2.53 mg m-2 (Figure 2). The intercept yielded Bads ) 8.6 ( 2.7 × 106 dm3 mol-1 for the holo-type and 3.4 ( 0.9 × 106 dm3 mol-1 for the apo-type. Using these values, the isotherm for the adsorption of R-LA onto the Pt surface at 313 K was calculated and plotted according to eq 4 (see inset in Figure 3). A good agreement between the experimental and simulated values confirmed the applicability of the Langmuir isotherm in the description of the adsorption of both types of R-LA onto the Pt surface. The parameter Bads, which reflects the affinity of the adsorbate molecules toward adsorption sites at a constant temperature, could be presented as52

Bads )

(

)

-∆Gads 1 exp 55.5 RT

(6)

where R (J mol-1 K-1) is the gas constant, T (K) is the temperature, ∆Gads (J mol-1) is the Gibbs free energy of adsorption, and 55.5 is the molar concentration of the water (mol dm-3), which is used as a solvent. Using this equation, the Gibbs free energy of adsorption of holo-R(52) Gomma, G. K.; Wahdan, M. H. Mater. Chem. Phys. 1994, 39, 142.

Figure 4. Dependence of the Gibbs free energy of adsorption on the temperature for (O, s) holo- and (4, - - -) apo-R-LA adsorbed onto Pt in phosphate buffer, pH 7.

LA onto the Pt surface in phosphate buffer solution at 313 K was calculated to be -52 ( 1 kJ mol-1 and a value of -49.6 ( 0.8 kJ mol-1 was calculated for the apo-type. Such high values indicate strong adsorption of R-LA onto the Pt surface via chemisorption. Using the data for the adsorption of apo-R-LA onto a hydrophobic silicon surface at pH 7 and 300 K, reported by Suttiprasit et al.,40 we calculated an adsorption Gibbs free energy value of ∼-39 kJ mol-1, and from the data for the adsorption of the protein onto a stainless steel 304 surface at 298 K and pH 6, presented by Addesso et al.,3 a calculated value was ∼-40 kJ mol-1. The somewhat higher values obtained in our case indicate a higher affinity of the protein toward adsorption onto a positively charged Pt surface. Figure 4 shows the dependence of the Gibbs free energy of adsorption on temperature for both types of protein at pH 7. With increase in temperature, the negative Gibbs free energy value increases with the linear dependence. The slope of the line for holo-R-LA yielded the value of entropy ∆Sads ) 197 ( 3 J mol-1 K-1 and the enthalpy of adsorption was calculated to be ∆Hads ) 12 ( 1 kJ mol-1. For apo-R-LA, the calculated values are ∆Sads ) 164 ( 3 J mol-1 K-1 and ∆Hads ) 2 ( 1 kJ mol-1. Positive ∆Hads values show that the adsorption of R-LA onto the Pt surface is an endothermic process. Haynes et al.48 showed that the enthalpy of adsorption of protein is a sum of individual enthalpies related to (i) changes in the state of hydration of the sorbent surface, ∆Hhyd, (ii) association/dissociation of protons with charged groups on the protein surface, ∆HH+, (iii) overlap of electric fields, ∆Hel, (iv) contribution to the incorporation of ions (other than protons) in the adsorbed layer, ∆Hion, and (v) structural rearrangement in the protein molecule, ∆Hstr.pr.. Therefore, a value of the adsorption enthalpy ∆Hads depends on the contribution of each of the mentioned individual enthalpies48

∆Hads ) ∆Hhyd + ∆HH+ + ∆Hel + ∆Hion + ∆Hstr.pr. (7) Since the ∆Hads values are relatively small (12 kJ mol-1 for the holo-type and 2 kJ mol-1 for the apo-type), the enthalpies on the right-hand side of the equation mostly cancel. From the result presented here, it is not possible to determine the enthalpy value of each individual subprocess, but Haynes et al.48 showed that values of

Adsorption of R-Lactalbumin at Pt+

∆Hstr.pr. are large and endothermic because of the loss of favorable intramolecular interactions within the protein when it adsorbs and unfolds on the sorbent surface (the transition enthalpy for thermal denaturation of apo-RLA at pH 7 in the presence of 0.5 M Na+ is ∆H ) 265 kJ mol-1 53) and values of ∆Hion are also large but exothermic because of the water-water hydrogen bond formation which accompanies the transfer of ions (phosphate anions in our case) from water to the adsorbed layer. The enthalpy changes associated with the remaining subprocesses are predicted to be relatively small but together could make a significant contribution to the sign and magnitude of ∆Hads.48 The small difference in the adsorption enthalpy values between the holo- and the apo-type confirms our previous conclusion that there is no adsorption-induced transition from the holo- to the apo-type, since the enthalpy value associated with the release of calcium is significantly higher (145 kJ mol-1 at pH 7.4 and 298 K18) than the difference observed in our case (10 kJ mol-1). In addition, the lower ∆Hads value obtained for the apo-type might come from the fact that apo-R-LA is in a molten globule state and already partially unfolded, and therefore its ∆Hstr.pr. value is lower in comparison to the values for the holo-type. Interfacial tension measurements performed by Matsumura et al.41 also showed that R-LA in the molten globule state is more easily unfolded at liquid interfaces than is the native protein. The small positive ∆Hads value indicates that the netinfluence of enthalpy on the adsorption of R-LA on Pt is minor under the experimental conditions applied in our experiments. However, from the calculated thermodynamic values it is apparent that the gain in entropy actually represents the driving force for the adsorption of R-LA onto the platinum surface, since the T∆Sads product ranges from 54 to 70 kJ mol-1 for the holo-type and 45 to 58 kJ mol-1 for the apo-type, depending on the temperature, which is considerably higher than the enthalpy values (12 and 2 kJ mol-1, respectively). The structure and thus the dimension of the molecule change when the protein adsorbs onto a surface. Hence, the induced structural changes can lead to a considerable entropy gain, which might be, as we see here, the driving force for adsorption. It has been shown in the literature48,54-56 that proteins with low native-state stabilities, such as R-LA, possess a strong driving force for adsorption related to breakdown of native tertiary and partially secondary structure; in other words, adsorption is driven by an increase in the conformational entropy of the protein. Large positive entropy values could arise from the unfolding of protein molecules upon adsorption. It has been shown57 that the increased rotational freedom of the polypeptide backbone which results from the complete unfolding of a native protein will lead to an entropy gain of 10 to 100 J K-1 per mole of amino acid residue. However, from the enthalpy and entropy values obtained in our case, one can conclude that R-LA does not undergo complete unfolding upon adsorption at the Pt surface. Measurements at pH 2. There have been many studies comparing the molten globule states which occur under various conditions, since the molten globule state is more of a continuum of conformational states with a varying degree of disorder found in the tertiary structure. There (53) Relkin, P. Crit. Rev. Food Sci. Nutr. 1996, 36, 565. (54) Norde, W.; Favier, J. P. Colloids Surf. 1992, 64, 87. (55) Van Wagenen, R. A.; Rockhold, S.; Andrade, J. D. Adv. Chem. Ser. 1982, 199, 351. (56) Bentaleb, A.; Abele, A.; Haikel, Y.; Schaaf, P.; Voegel, J. C. Langmuir 1998, 14, 6493. (57) DeWit, J. N.; Klarenbeek, G. J. Dairy Sci. 1984, 67, 2701.

Langmuir, Vol. 16, No. 22, 2000 8485

Figure 5. Surface charge density as a function of concentration of apo-R-LA in sulfuric acid, pH 2: (b) 273; (O) 299; (1) 313; (3) 323; (9) 333; (0) 343; and (() 353 K.

is much debate over whether the molten globule state due to extreme pH is equivalent to the molten globule state occurring upon the removal of the calcium ion at neutral pH. It is possible to examine the surface concentrations at different temperatures and pH levels to see if comparisons can be made regarding what is known about the conformational changes of the proteins and the shape of these graphs. The surface charge density values at various concentrations of R-LA over a temperature range of 273 K to 353 K at pH 2 were measured with an anodic end potential which normally corresponds to a monolayer of adsorbed OH species on the Pt surface at these temperatures. As an example, the dependence of the adsorption charge density of apo-R-LA on the equilibrium protein concentration in the bulk solution is presented in Figure 5. With an increase in protein concentration in the bulk solution, the surface charge density values sharply increase and reach their maximum at a very low concentration of the protein in the bulk solution, above which they slightly decrease and level off into the plateaus. The rising portions of the presented curves could be explained in a similar manner as those in Figure 2. However, the decrease in the surface charge density after a maximum value (Figure 5) indicates that the protein either changes its conformation or orientation after a “critical” equilibrium surface concentration at a certain temperature is reached or partially desorbs, as suggested by Soderquist and Walton.58 They concluded that, if given sufficient time, adsorbed proteins undergo conformational changes which lead to increased surface interaction. During this process, proteins less optimally adsorbed undergo desorption, hence the observed “overshoot” in the curve at lower protein concentrations in the bulk solution (Figure 5). Van Dulm et al.59 (in a study of human plasma albumin on polystyrene lattices) showed that fast initial adsorption was followed by desorption, probably because of conformational changes of the adsorbed protein, which induced the release of less tightly bound protein. This result was observed at pH 4, where the albumin has a net positive charge and was not observed at pH 7.4, where it is highly negative. A similar explanation may be applied to our results. With increase in temperature, the plateau surface charge value obtained at pH 2 increases, which is clearly (58) Soderquist, M. E.; Walton A. G. J. Colloid Interface Sci. 1980, 75, 386. (59) Van Dulm, P.; Norde, W. J. Colloid Interface Sci. 1983, 91, 248.

8486

Langmuir, Vol. 16, No. 22, 2000

Figure 6. Plateau surface concentration of (O) holo- and (4) apo-R-LA at various temperatures obtained in sulfuric acid, pH 2, and phosphate buffer, pH 11.

presented in Figure 6. The plot shows that both types of the protein behave almost similar at pH 2 in the whole temperature range investigated, which is quite different than the situation at pH 7 (Figure 3). This indicates that, at pH 2, the protein exists mainly as an apo-type of R-LA (i.e., the protein is significantly depleted in calcium). Griko et al.18 showed that, with decrease in pH, the thermal stability of R-LA rapidly decreases below pH 5.2 because of the decrease in Ca2+ binding affinity. At pH 3, the transition temperature is below 288 K, and log Ka of Ca2+ association dropped from ∼6.2 at pH 7 down to ∼2.2 at pH 3. By extrapolation of their values to pH 2, it appears that, at 273 K, the protein is completely depleted in calcium, which agrees with the conclusion obtained from our results (Figure 6). The calcium ion is known to help stabilize the molten globule state at low pH but only in moderate calcium concentrations (e.g., 2 mM),46 which was not the case in these experiments. The lack of calcium ions present in the electrolyte solution probably increased the release of the bound calcium in the holo-type and therefore increased the resemblance in the conformation of both types of proteins at this pH. If we compare the plateau surface charge values obtained at pH 2 (Figure 6) to those at pH 7 (Figure 3), we will see that the values at pH 2 are considerably lower. This is the result of conformational changes occurring when lowering pH below 314 (the acid conformational transition). Below pH 3, R-LA, which is transformed into a molten globule state, occupies a larger volume than the native protein. Consequently, the number of molecules that occupy a unit area of the Pt surface is smaller. Figure 7 shows that the dependence of the Gibbs free energy of adsorption on temperature at pH 2 is linear for both types of protein. The slope of the line for holo-R-LA yielded the value of entropy ∆Sads ) 258 ( 1 J mol-1K-1, and the enthalpy of adsorption was calculated to be ∆Hads ) 25.1 ( 0.3 kJ mol-1. For apo-R-LA, the calculated values are ∆Sads ) 240 ( 1 J mol-1K-1 and ∆Hads ) 19.7 ( 0.2 kJ mol-1. The difference between the enthalpy and entropy values of the two types of protein is much smaller than that obtained at pH 7, again suggesting that the holotype is mainly depleted in calcium at pH 2. The enthalpy values obtained at pH 2 are higher than those obtained at pH 7. However, again the gain in entropy actually represents the driving force for the adsorption of R-LA onto the platinum surface at pH 2, since the T∆Sads product ranges from 70 to 91 kJ mol-1 for the holo-type

Cabilio et al.

Figure 7. Dependence of the Gibbs free energy of adsorption on the temperature for (O, s) holo- and (4, - - -) apo-R-LA adsorbed onto Pt in sulfuric acid, pH 2. Inset shows the dependence of the adsorption affinity constant for the apo-type on pH at (3) 273 and (0) 299 K.

Figure 8. Surface charge density as a function of concentration of holo-R-LA in phosphate buffer, pH 11: (b) 273; (O) 299; (1) 313; and (3) 323 K. Inset: dependence of the Gibbs free energy of adsorption on the temperature for (O, s) holo- and (4, - - -) apo-R-LA adsorbed onto Pt.

and 66 to 85 kJ mol-1 for the apo-type, depending on the temperature, which is considerably higher than the enthalpy values (25.1 and 19.7 kJ mol-1, respectively). Measurements at pH 11. The series of experiments performed at pH 11 are unique in behavior in the fact that two plateau levels were obtained on the isotherms (see Figure 8). This suggests that the protein changes in conformation or in orientation after a certain surface concentration (which is in equilibrium with the protein concentration in the bulk solution) is reached to allow more protein to adsorb onto the electrode. The existence of the stepped isotherm (Figure 8) can be explained by adapting Fair and Jamieson’s60 approach: at low concentrations (first plateau), adsorption proceeds by a random uncorrelated mode, resulting in a completely disorganized structure, containing approximately 75-80% /Q2.plateau ). The adsorbed protein occupied area (Q1.plateau ads ads has sufficient time and room to acclimate to its new microenvironment by conformational changes. At concentrations higher than the concentration corresponding to the end of the first plateau, the surface free energy of the protein layer favors a transition from the less dense (60) Fair, B. D.; Jamieson, A. M. J. Colloid Interface Sci. 1980, 77, 525.

Adsorption of R-Lactalbumin at Pt+

random layer to a more dense, partly ordered structure, whose degree of order is kinetically limited by the configurational relaxation time at the surface. However, the collision frequency of the protein molecules with the surface is still too low to maintain growth of the more “ordered” structure. At a concentration that corresponds to the beginning of the second plateau (“critical bulk concentration”), the collision frequency is sufficiently large to overcome the kinetic limitation, and the adsorbed protein has neither the time nor the room to optimize its interaction with the surface. At this point, a transition to a cooperative adsorption mode occurs, in which a closepacked more “ordered” surface phase of the protein is formed. Thus, the “critical bulk concentration” may be regarded as a concentration at which the average time between consecutive collisions of the protein molecules at a growth site on the surface becomes comparable to the configurational relaxation time at the surface. Morrissey61 also suggested a similar explanation of the appearance of stepped isotherms. Stepped isotherms were reported by Galisteo et al.42 in their study of the adsorption of R-LA onto AgI sol at pH 7, using a colorimetric Lowry method, and by Bentaleb et al.62 in the study of adsorption of R-LA onto a titanium oxide surface at pH 7.5, using radiolabeling by 125I. Using cyclic voltammetry and electrochemical impedance spectroscopy techniques, Jackson et al.27 reported stepped isotherms in their study of adsorption of BSA onto a Ti surface at pH 7.4. The same was also reported by Fair et al.60 for the adsorption of BSA and IgG onto a polysterene surface at pH 7, using a solutiondepletion method. Like the proteins at pH 2, the difference between the plateau surface concentrations of the apo- and holo-type at a given temperature at pH 11 is negligible (Figure 6), probably because of the release of calcium in the holotype. However, the plateau surface concentration values reached at this pH are slightly higher than those reached at pH 2 but still lower than those reached under neutral conditions for either type of protein (Figure 3). Since the molecule has high positive charge at pH 2 and high negative charge at pH 1142 and the Pt surface is positively charged, the observed behavior suggests that the adsorption of R-LA onto the Pt surface is also partially controlled by electrostatic repulsion between the protein molecules at higher concentrations. However, at the same time, the affinity of the protein to adsorbs onto the Pt surface at pH 11 is considerably lower than that at pH 2 and pH 7 (see inset in Figure 7), even though the protein has a large negative net charge at pH 11 and the surface is positively charged (compare the values in the inset in Figure 7). In contrast, at pH 2, the protein has a large positive charge but yet expresses the highest affinity toward adsorption at low concentrations in the bulk solution. One possible explanation for these contradictions could be as follows; at pH 2 the carboxyl groups in R-LA are protonated and the interaction between (solvated) carboxyl groups and water molecules is rather weak (a dipole-dipole type of interaction). On the other hand, at pH 11, carboxyl groups are deprotonated and negatively charged. Their interaction with water molecules is much stronger (ion-dipole interaction) than that at pH 2. Consequently, dehydration of carboxylate groups at pH 11 and their subsequent attachment to the Pt surface require more energy and thus result in a smaller affinity of the protein toward adsorption at the Pt surface. (61) Morrissey, B. W. Ann. N. Y. Acad. Sci. 1977, 288, 50. (62) Bentaleb, A.; Haı¨kel, Y.; Voegel, J. C.; Schaaf, P. J. Biomed. Mater. Res. 1998, 40, 449.

Langmuir, Vol. 16, No. 22, 2000 8487

In addition, solvation of protein molecules and the Pt surface greatly minimizes a repelling force between the positively charged protein and the positively charged Pt surface at pH 2 but, at the same time, also minimizes an attraction force at pH 11. To calculate ∆Gads values for R-LA at pH 11, only the lower concentration region was used (first plateau). The dependence of the Gibbs free energy of adsorption on temperature was found to be linear (see inset in Figure 8), and from the slope of the lines the entropy for the adsorption of holo-type was calculated to be 207 ( 6 J mol-1 K-1 and the intercept yielded ∆Hads ) 16 ( 2 kJ mol-1, while, for the adsorption of apo-type, the entropy value, ∆Sads, was 167 ( 3 J mol-1 K-1 and the enthalpy was 3.8 ( 0.6 kJ mol-1. If we compare these values to the values obtained at pH 2 and pH 7, we will see that they follow the trend already noticed with the surface concentration values (i.e., they are between the values obtained at pH 2 and pH 7 but closer to the values obtained at pH 7). Similar to the situation at pH 2 and pH 7, a driving force for the adsorption of R-LA onto the Pt surface at pH 11 again represents the change in entropy, since the product T∆Sads is much higher than the enthalpy of adsorption ∆Hads for both types of protein. Conclusions The interfacial behavior of holo- and apo-R-LA at a Pt surface was studied over the temperature range 273 to 353 K in an acidic, neutral, and alkaline medium, using the cyclic voltammetry technique. It was shown that the surface charge density, resulting from protein adsorption, is directly proportional to the amount of adsorbed protein (surface concentration), indicating that adsorption at anodic potentials is accompanied by the transfer of charge (i.e., chemisorption through carboxylate groups on the protein). The adsorption isotherms obtained at pH 2 showed very high affinity of the protein toward adsorption onto a Pt surface, with an indication of either conformational/ orientation changes in the protein or its partial desorption at very low equilibrium concentrations in the bulk solution. On the other hand, the isotherms at pH 7 were of a “normal” Langmuirian type, resulting in a single saturation plateau, while the isotherms at pH 11 resulted in a double plateau. The highest saturated surface concentration was obtained at pH 7, indicating that the protein is the most compact at this pH. The difference in the saturation concentration between the two types of protein was observed in the whole temperature range and was explained on the basis of the presence/absence of calcium ion in the molecule (existence of a molten globule state). At pH 2 and pH 11, lower saturated surface concentration values were obtained and no difference in the amount of adsorbed protein was noticed between holo- and apo-type proteins. This suggests that, at pH 2 and pH 11, only the calcium-free molten globule state of the protein exists. The adsorption process was described with the Langmuir adsorption isotherm. From the calculated Gibbs free energies of adsorption it was concluded that R-LA molecules strongly adsorb onto the Pt surface via chemisorption. The adsorption process was found to be slightly endothermic, presumably resulting from the excess energetics required for the breaking of intramolecular interactions relative to those involved in the formation of protein-metal bonds. The adsorption of R-LA onto a Pt surface was found to be an entropically governed process,

8488

Langmuir, Vol. 16, No. 22, 2000

also suggesting structural unfolding of the protein at the electrode surface. Therefore, the evidence from the present work suggests that disruption of tertiary structure of the protein molecule occurs upon adsorption at the Pt surface and that the breaking of intramolecular interactions during the adsorption governs the rate of the process.

Cabilio et al.

Acknowledgment. Grateful acknowledgment is made to the Dairy Farmers of Canada and the Natural Science and Engineering Research Council of Canada for support of this research. LA0003948