Thermodynamic Properties of the Complex Formed by Interaction of

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Thermodynamic Properties of the Complex Formed by Interaction of Two Anionic Amphiphilic Penicillins with Human Serum Albumin Silvia Barbosa,† Pablo Taboada,† David Attwood,‡ and Vı´ctor Mosquera*,† Grupo de Fı´sica de Coloides y Polı´meros, Departamento de Fı´sica de la Materia Condensada, Facultad de Fı´sica, Universidad de Santiago de Compostela, Spain and School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester M13 9PL, United Kingdom Received June 23, 2003. In Final Form: September 5, 2003 The partial specific volume and adiabatic compressibility of the complexes formed by the interaction of the amphiphilic penicillins cloxacillin and dicloxacillin with human serum albumin at a concentration of 0.2% w/v in aqueous solution, and at penicillin concentrations below the critical micellar concentration, were obtained from density and sound velocity measurements at temperatures of 15, 25, and 35 °C. Increases of the apparent partial specific volumes and adiabatic compressibilities of the drug-protein complexes with drug concentration in the initial stages of drug adsorption have been discussed before in terms of the development of a diffuse double layer around the complex. Subsequent decreases in these quantities after saturation of the protein surface at higher drug concentrations are thought to reflex conformational changes in the protein attributable to a more compacted tertiary structure and a possible onset of premicellar aggregation of drug in solution causing dehydration of the complex because of competition for available free water. Binding isotherms obtained by the application of isothermal titration calorimetry to a study of the adsorption process at 25 °C have provided support for these conclusions.

1. Introduction Investigation of the mechanism by which penicillins bind to serum albumin is indispensable for understanding the transport functions of serum albumin and for obtaining quantitative characteristics of this interaction for practical purposes. Albumin is capable of reversibly binding many substances and can therefore assume transport and vehicle functions in the body. The ability of the albumin to bind several anions and cations can be exploited in studies of ion binding. The affinity of albumin for ligands depends on the hydrophobic character of the molecules and their charge. Molecules with long alkyl chains and negatively charged groups are strongly bound, while molecules with short chains and positively charged groups are bound less firmly.1 Human serum albumin (HSA) acts as a carrier for several amphiphilic drugs from bloodstream to tissues. Investigation of the volumetric and compressibility data of the complexes formed by the interaction of amphiphilic drugs and the protein is important for the construction from genetic engineering of de novo stable proteins, in which correct chain packing is crucial.2 Studies on the structures and interactions of the complexes formed between proteins and surfactants in aqueous solutions have been extensively reviewed.3,4 Protein-surfactant complexes can be conveniently divided into three main categories:5 (i) polyelectrolytes and charged * To whom correspondence should be addressed. E-mail: [email protected]; tel: 0034981563100 ext.14056; fax: 0034981520676. † Universidad de Santiago de Compostela. ‡ University of Manchester. (1) Peters, T. J. All about Albumin Biochemistry, Genetics, and Medical Applications; Academic Press: San Diego, CA, 1996. (2) Harbury, P. B.; Plees, J. C.; Tidor, B.; Alber, T.; Kim, S. Science 1998, 282, 1462. (3) Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes; Wiley-Interscience: New York, 1980. (4) Goddard, E. D.; Ananthapadmanabhan, K. P. Interactions of Surfactants with Polymers and Proteins; CRC Press: London, 1993.

ionic amphiphilic systems, where the protein and the amphiphilic molecule can have opposite charge, giving rise to Coulombic and hydrophobic interactions, or the same charge (as in the case of the present work), where the interaction is primarily hydrophobic; (ii) neutral polymer and ionic amphiphilic systems; and (iii) less common kinds of systems, containing either a polyelectrolyte and a nonionic amphiphile or two neutral species. The globular protein serum albumin, chosen for this study, has been widely used as a model protein for studying the interaction between proteins and different surface substrates. HSA consists of 586 amino acids in a single polypeptide chain with a molar mass of 66411 g mol-1. The serum albumin molecule is a single polypeptide chain folded into a tertiary globular conformation developing three domains in the molecule.1 The amino acid sequence in the chain is nearly repetitive so that the domains have almost similar sequence and structure; the backbone is interlinked through 17 disulfide bonds which, in general, are known for producing an overall rigidity in the globular structure. The surface of the protein molecule in contact with aqueous solvent comprises hydrophilic and hydrophobic groups in almost equal number.6 The apparent partial specific volume, v, and adiabatic compressibility, βS, of the protein are important physical quantities directly related to the compactness or globularity of the protein molecule because they involve the contributions of surface hydration and internal cavity.7,8 The hydrophobic groups of the protein are assumed to be surrounded by voluminous icelike water structures. Transfer of the hydrophobic groups into an aqueous solvent will lead to two different volume effects: (1) expansion due to formation of icelike (5) Diamant, H.; Andelman, D. Europhys. Lett. 1999, 48, 170. (6) Richards, F. M. Annu. Rev. Biophys. Bioeng. 1977, 6, 151. (7) Scharade, P.; Klein, H.; Egry, I.; Ademovic, Z.; Klee D. J. Colloid Interface Sci. 2001, 234, 445. (8) Nemethy, G.; Scheraga, H. A. J. Chem. Phys. 1962, 36, 3401.

10.1021/la035106x CCC: $25.00 © 2003 American Chemical Society Published on Web 10/17/2003

Thermodynamic Properties Scheme 1

structures9 in the neighborhood of the hydrophobic groups, (2) shrinkage due to the intrusion of the hydrophobic groups into empty spaces inherent in the water structure. Sudlow and co-workers10 have demonstrated that most drugs bind with high affinity to two main regions identified from the crystal structure of the protein bound to different molecules,11 referred to as Site I or warfarin site and Site II or the indole-benzodiazepine site. Site II is a hydrophobic cleft about 12-16 Å deep and about 8 Å wide with a cationic group located near the protein surface. Penicillin molecules might bind in this site when binding starts to be extensive, that is, in the cooperative region.12-15 The hydrophobic nature of penicillins as demonstrated by their selfassociation behavior15,16 and the evidence of hydrophobic cavities in the structure of bovine and human serum albumin17,18 suggest that noncovalent binding of penicillins onto serum albumin takes place. Our previous studies of the solution properties of a large number of penicillins19-23 have characterized the selfassembly in aqueous solution as a function of electrolyte content and temperature. Cloxacillin and dicloxacillin are structurally similar, differing only in an additional chlorine atom on the phenyl ring of dicloxacillin (see Scheme 1). They are anionic molecules with pKa’s of 2.7 and 2.8, respectively, and are fully ionized in water. Static light scattering and NMR studies19 have shown that both drugs form small aggregates (typically five to six molecules) at a well-defined critical micellar concentration (cmc) in aqueous solution. Compressibility is a novel measure of the structural flexibility of HSA in solution since it is directly linked to its volume change, which gives a visual approach to the protein dynamics.24 The adiabatic compressibility is a macroscopic quantity involving contributions from both surface hydration and imperfect atomic packing in the internal cavity and is known to sensitively reflect the characteristic structures of native proteins25,26 and the (9) Neal, J. L.; Goring, D. A. J. Phys. Chem. 1970, 74, 658. (10) Sudlow, G.; Birkett, D. J.; Wade, D. N. Mol. Pharmacol. 1975, 11, 824. (11) Diamant, H.; Andelman, D. Phys. Rev. E 2000, 61, 6740. (12) Chadborn, N.; Bryant, J.; Bain, A. J.; O’Shea, P. Biophys. J. 1999, 76, 2198. (13) Gelamo, E. L.; Tabak, M. Spectrochim. Acta A 2000, 56, 2256. (14) Joos, R. W.; Hall, W. H. J. Pharmacol. Exp. Ther. 1969, 166, 113. (15) Attwood, D.; Agarwal S. P. J. Pharm. Pharmacol. 1984, 36, 563. (16) Taboada, P.; Attwood, D.; Ruso, J. M.; Sarmiento, F.; Mosquera, V. Langmuir 1999, 15, 2022. (17) Carter, D. C.; He, X. M. Science 1990, 249, 302. (18) He, X. M.; Carter, D. C. Nature 1992, 358, 209. (19) Taboada, P.; Attwood, D.; Garcı´a, M.; Jones, M. N.; Ruso, J. M.; Mosquera, V.; Sarmiento, F. J. Colloid Interface Sci. 2000, 221, 242. (20) Taboada, P.; Attwood, D.; Ruso, J. M.; Garcı´a, M.; Sarmiento, F.; Mosquera, V. J. Colloid Interface Sci. 1999, 216, 270. (21) Varela, L. M.; Rega, C.; Sua´rez-Filloy, M. J.; Ruso, J. M.; Prieto, G.; Attwood, D.; Sarmiento, F.; Mosquera, V. Langmuir 1999, 15, 6285. (22) Taboada, P.; Attwood, D.; Ruso, J. M.; Garcı´a, M.; Sarmiento, F.; Mosquera, V. J. Colloid Interface Sci. 1999, 220, 288. (23) Ruso, J. M.; Attwood, D.; Garcı´a, M.; Taboada, P.; Varela, L. M.; Mosquera, V. Langmuir 2001, 17, 5189. (24) Cooper, A. Thermodynamic fluctuations in protein molecules. Proc. Natl. Acad. Sci. U.S.A. 1976. (25) Gekko, K.; Noguchi, H. J. Phys. Chem. 1979, 83, 2706.

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conformational changes induced by denaturation.27 In an amphiphilic molecule dissolved in water, reorganization of the water structure results in a negative compressibility. The formation of the complex between the ligand and the polymer involves changes in the hydration water of both host and guest molecules that are reflected in thermodynamic properties related to the volume and compressibility of the implicated species. The changes of compressibility of proteins because of ligand binding should shed light on how conformational flexibility manifests its effect on the kinetic reaction through the changes in internal cavities and solvent-accessible surface area. We have previously reported the apparent partial specific volume, v, and the apparent partial specific adiabatic compressibility of the protein, βS, at a fixed concentration of 2 g dm-3 of HSA as a function of the concentration of the penicillin drugs cloxacillin and dicloxacillin and at the temperature of 30 °C. For this globular protein, it has been previously shown by Gekko et al.28,29 that the protein concentration dependences of v and βS are negligible at concentrations up to 5 g dm-3. The results provided information of how the changes in the volumetric and compressibility data can be ascribed to the protein-penicillin complex formation. The data suggested that the adsorption process can be divided into two distinct steps for both penicillins, with an initial pronounced increase of the volume and compressibility with penicillin concentration that reflects the dehydration of each solute molecule and a subsequent decrease due to conformational changes in the protein that could be attributed to a more compacted structure. In the present work, we aimed to complete the thermodynamic characterization of the complex, in the same range of concentrations, determining the volumetric and compressibility data at the range of temperatures 15-35 °C to obtain a comprehensive thermodynamic description of complex formation in aqueous solution in relation to the protein structural behavior. In addition, the binding isotherm for complex formation has been determined by isothermal titration calorimetry at the temperature of 25 °C. 2. Experimental Section 2.1. Materials and Methods. Human serum albumin (7002490-7, 98% purity), sodium cloxacillin monohydrate ([5-methyl3-(o-chlorophenyl)-4-isoxazolyl]penicillin), and sodium dicloxacillin monohydrate ([3-(2,6-dichlorophenyl)-5-methyl-4-isoxazolyl] penicillin) were obtained from Sigma Chemical Company. Experiments were carried out at 15, 25, and 35 °C using doubledistilled, deionized, and degassed water. All the samples were prepared below the critical micellar concentration of the penicillins, well below the solubility limit, using a Metler AT20 balance with a precision of (0.001 mg to weigh the components. The samples were stored in sealed glass containers and transferred without delay to the densitometer-ultrasound measurement cell by means of glass syringes. Each set of density-ultrasound measurements was carried out at least three times, the results averaged, and the value then used to calculate the apparent specific volume, v, and the ultrasound velocity of the complex. 2.2. Adsorption of Cloxacillin and Dicloxacillin onto Albumin. Aliquots of 2.5 cm3 of a 0.4% (w/v) of HSA were added to equal volumes of aqueous solutions of penicillin of known concentration to give a final solution in which the concentration of HSA was 0.2% (w/v). The concentrations of both penicillins were always below the critical micelle concentration, (0.047 g cm-3 for both penicillins). (26) Chalikian, T. V.; Totrov, M.; Abagyan, R.; Breslauer, K. J. J. Mol. Biol. 1996, 260, 588. (27) Kharakoz, D. P. Biochemistry 1997, 36, 10276. (28) Gekko, K.; Hasegawa, Y. Biochemistry 1986, 25, 6563. (29) Gekko, K.; Hasegawa, Y. J. Phys. Chem. 1989, 93, 426.

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2.3. Density and Ultrasound Velocity Measurements. Density and ultrasound velocity measurements were carried out using an Anton Paar DSA 5000 densimeter and sound velocity analyzer. One of the principal limitations of custom-built systems resides in possible temperature drifts. This problem was circumvented in the present study by maintaining the temperature control by the Peltier effect giving a resolution of (0.001 K and uncertainties in density of ca. (1 × 10-6 g cm-3. Errors in ultrasound velocity measurements also arise mainly from variations of temperature and in this study the resolution was (0.01 m s-1. The experimental procedures were essentially the same as those used in the previous study.30 For a nonscattering system, there is a simple relationship between the ultrasonic velocity of a solution and its physical properties. Assuming that the wavelength of sound is much greater than the particle size and independent of the frequency, the adiabatic compressibilities of sample solution, β, and solvent, β0, can be calculated using the Laplace equation, β ) 1/(Fu2) with a sound velocity, u, and density, F, data set of the sample solution. For systems where scattering is important, the velocity is dependent on the particle size and frequency and appreciable velocity dispersion may occur. The apparent partial specific volume, v, and the apparent partial specific adiabatic compressibility, βS, of HSA and its ligand complexes were calculated, using eqs 1 and 2 with u0 as solvent sound velocity and F0 as solvent density and at the temperatures of 15, 25, and 35 °C

v)

βS ) -

[

]

1 F-c 1c F0

( ) ( )[

1 ∂v v ∂p

)

S

(1)

]

β0 β F-c vc β0 F0

(2)

where p is the pressure and c the concentration of the solute (protein) in g cm-3. 2.4 Calorimetry. Heats of dilution were measured using a VP-ITC titration microcalorimeter (MicroCal Inc., Northampton, MA). Small aliquots of stock solution of penicillin at a concentration above the cmc were injected into a known volume of a 0.2% (w/v) aqueous solution of HSA (ca. 1 cm3) held in the cell of the calorimeter, initially to produce solution below the cmc. Repeated additions of the stock solution gave the heat evolved (Q) as a function of penicillin concentration. Correction of this heat of dilution for the heats of dilution of HSA and the penicillins gave a value of Q representing only the heat of binding the penicillins to the biopolymer. In the experiment, a stock solution of concentration 17 g dm-3 was added at intervals of 300 s using 28 injections each of 10 mm3.

3. Results and Discussion The apparent partial specific volume, v, and adiabatic compressibility, βS, of HSA and its ligand complexes are macroscopic quantities which are particularly sensitive to the hydration properties of solvent-exposed atomic groups, as well as the structure, dynamics, and conformational properties of the solvent-inaccessible protein interior.26,31 The HSA-penicillin complex formation may be thought of as proceeding in three states:32 (a) hydrophobic interaction between the polymer and the ligand, (b) changes in hydration of the interacting molecules, and (c) conformational changes in the polymer. These processes should manifest themselves in the volumetric and compressibility properties via changes in internal atomic packing and surface hydration of the polymer. The data reported here are for concentrations below the critical micelle concentrations of both penicillins. (30) Gutie´rrez-Pichel, M.; Taboada, P.; Varela, L. M.; Attwood, D.; Mosquera, V. Langmuir 2002, 18, 3650. (31) Chalikian, T. V.; Sarvazyan, A. P.; Breslauer, K. J. Biophys. Chem. 1994, 51, 89. (32) Kamiyama, T.; Gekko, K. Biochim. Biophys. Acta 2000, 1478, 257.

Critical aggregation concentrations in water and in the presence of 0.0625% (w/v) HSA of 0.098 mol kg-1 (0.047 g cm-3) and 0.136 mol kg-1 for cloxacillin and HSA/ cloxacillin and 0.093 mol kg-1 (0.047 g cm-3) and 0.114 for dicloxacillin and HSA/dicloxacillin, respectively, were obtained in a previous work.33 3.1. Volume and Compressibility Changes. The apparent partial specific volume of a protein in water, v, can be considered to be the sum of the following terms:34

v ) va + vc + ∆vh + βT0 RT

(3)

where va is the constitutive atomic volume and vc is a contribution arising from imperfect atomic packing in the internal cavity. ∆vh is the volume change due to hydration and represents the change in the solvent volume associated with interactions of water molecules with charged (electrostriction) and polar (hydrogen bonding) atomic groups of the protein and hydrophobic hydration around the nonpolar groups. Each of these interactions produce a negative volume change, and hence ∆vh is normally negative. βT0 is the coefficient of isothermal compressibility of the solvent, R is the universal gas constant, and T is the absolute temperature. The last term, βT0 RT, describes the volume effect related to the kinetic contribution to the pressure of a solute molecule because of the translational degrees of freedom.35 The value of this term is about 1 cm3 mol-1 and usually can be ignored when considering large solutes such as proteins. Our results show that there is a volume variation for both penicillins of ∼0.05 cm3 g-1 that can be attributed to the complex formation. There is thought to be very little or no volume difference between the aqueous unfolded conformation and the refolded state of proteins.36 This result has been referred to as the protein volume paradox and has been interpreted on the basis of the thermal volume concept, which is related to volume changes for protein unfolding involving changes in the solvent-accessible surface area. In a previous work, we have calculated the penicillins apparent molal volumes at infinite dilution37 (≈0.64 cm3/g for both penicillins) that can be identified with the partial molal volumes of the hydrated monomers. The apparent specific volumes of the biopolymer obtained in this work are, in general, in good agreement with the values previously reported by Iqbal and Verrel.38 Figure 1 shows the partial specific volume of HSApenicillin complexes as a function of penicillin concentration and at the temperatures of 15, 25, and 35 °C. As can be observed in the figure, the volumes of the complex are practically the same for both penicillins. The adsorption process suggested by these data can be divided into two distinct regions for both penicillins. There is an initial pronounced increase of the volume of the HSA-penicillin complex at drug concentrations up to approximately 0.012 g cm-3. It can be considered that at this concentration the protein binding sites are saturated. In the systems considered here, in addition to hydration and adsorption of the negative charged penicillin monomer as the solute concentration is increased, several additional parameters might contribute to the volume and compressibility of the (33) Taboada, P.; Attwood, D.; Ruso, J. M.; Sarmiento, F.; Mosquera, V. Langmuir 1999, 15, 2022. (34) Dubins, D. N.; Filfil, R.; Macgregor, R. B.; Chalikian, T. V. J. Phys. Chem. B 2000, 104, 390. (35) Stillinger, F. H. J. Solution Chem. 1973, 2, 141. (36) Valdez, D.; Le Hue´rou, J. Y.; Gindre, M.; Urbach, W.; Waks, M. Biophys. J. 2001, 80, 2751. (37) Taboada, P.; Gutierrez-Pichel, M.; Barbosa, S.; Attwood, D.; Mosquera, V. PCCP 2003, 5, 703. (38) Iqbal, M.; Verrall, R. E. J. Phys. Chem. 1987, 91, 1935.

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Figure 1. Apparent partial specific volume, v, of HSApenicillin complexes as function of penicillin concentration, c, for (a) dicloxacillin and (b) cloxacillin at (9) 15, (2) 25, and ([) 30 °C.

Figure 2. Apparent partial specific isentropic compressibility, βS, of HSA as function of concentration, c, of (a) dicloxacillin and (b) cloxacillin at (9) 15, (2) 25, and ([) 30 °C.

protein in the penicillin solution. The protein contains patches of anionic and cationic groups on its surface,39 being its net charge negative. So, the main specific protein-ligand interaction, between the anionic net charged biopolymer and the negatively charged penicillins, is of hydrophobic type that produces an anionic surface of the complex and a neutralizing diffuse double layer formed by cationic counterions, which contribute to an increase in both complex volume and compressibility. The solvent reorganization in the vicinity of the complex interface gives rise to the change of volume observed. The protein volume at penicillin concentrations greater than approximately 0.012 g cm-3 decreases with penicillin concentration, the volume reduction being more pronounced in the case of dicloxacillin where the volume observed at the highest drug concentration is lower than that of the protein in drug-free solution. A most likely cause of the observed decrease of volume of both drugprotein complexes is a change in the conformation of the protein, the tertiary structure becoming more compact as a consequence of drug binding. However, we consider that concomitant changes in the hydration of the drug-protein complex are occurring as a result of competition for available water at the surface of the complex because of self-association of the penicillins in solution at low drug concentrations. In a recent study, we have shown that as a consequence of nonelectrostatic penicillin-penicillin interactions such as hydrogen bonding, there is a weak association of monomers resulting in the formation of dimers and trimers (stacks) in aqueous solutions of these drugs at premicellar concentrations.30 The stacking process may be considered to involve the approach of two monomeric hydrophobic solutes from infinity to close contact, in such a way that only part of the hydration

sheath of both molecules is removed. The solvation of these small aggregates and solvent reorganization in the vicinity of the surface of the drug-protein complex may induce a perturbation of the chemical potential of the complex such that less water is available for the protein, which now has to compete for it. Under these conditions, complete hydration of the protein complex is not possible and the apparent partial specific volume decreases. Figure 1 shows a more gradual change of partial specific volume of the HSA/cloxacillin complex with added drug concentration with an apparent plateau in the region of drug saturation. Similar differences between the complexes formed with the two drugs are observed from ITC measurements, as discussed below. Figure 2 shows the apparent partial specific adiabatic compressibility of HSA-penicillin complexes as a function of penicillin concentration. The plots may be explained using an adsorption process similar to that used to describe the volumetric data. Since the constitutive atomic volume may be assumed to be incompressible, the apparent partial specific adiabatic compressibility of a protein, βS, is mainly determined by the internal cavity and surface hydration as follows:25,28

(39) Hattori, T.; Kimura, K.; Seyrek, E.; Dubin, P. L. Anal. Biochem. 2001, 295, 158.

( )( ) ( )(

βS ) -

1 ∂v v ∂p

)-

S

)

1 ∂vc ∂ (∆vh) + v ∂p ∂p

S

) βc + βh (4)

where βc ) -(1/v)(∂vc/∂p)S is the intrinsic compressibility of the protein molecule and reflects the imperfect packing of the polypeptide chains. For a globular protein, βc contributes positively to the adiabatic compressibility of the protein.32 βh ) -(1/v)(∂∆vh/∂p)S is the compressibility effect of hydration and reflects the decrease in the compressibility of the solvent resulting from interactions between the solute atomic groups and the surrounding

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Figure 3. ITC determination of the binding isotherms of HSA/penicillin as function of concentration, c, of (a) dicloxacillin and (b) cloxacillin at 25 °C.

water molecules.40 For a globular protein, βc contributes negatively to the adiabatic compressibility of the protein. The compressibility of the protein interior would not be expected to vary greatly because the interior packing density of normal globular proteins falls within a narrow range.41 Hence, the apparent partial specific isentropic compressibility of the protein, βS, is mainly determined by an increase due to the electric double layer formation and a subsequent decrease due to dehydration as discussed above. The differences in the plots for the two drug-protein systems are comparable to those noted in the volumetric results of Figure 1. 3.2. Calorimetry. Figure 3 shows the binding isotherms for the two HSA/penicillin systems. Region I of Figure 3b shows that a saturation binding enthalpy of ∼0.6 kJ mol-1 is achieved at a cloxacillin concentration (g cm-3) c ∼ 0.014, which is a consequence of adsorption of the negatively charged cloxacillin monomer within the hydrophobic cavities of the protein molecule.18 The plateau region (region II), ∆H ) 0 at concentrations (g cm-3) 0.014 e c e 0.022, may be attributed to the saturation of the protein sites, leading to region III where the exothermic enthalpy change may be a consequence of dehydration arising from competition for available water as a result of concomitant premicellar aggregation. Regions I and III of the binding isotherm for dicloxacillin, Figure 3a, are similar to those for cloxacillin binding, with a saturation binding enthalpy of ∼0.2 kJ mol-1, but this plot has no region equivalent to the plateau (region II) of Figure 3b. Thus, the results from the each of the experimental techniques of this study have shown evidence of a plateau region in the HSA-cloxacillin system that is not observ(40) Richards, F. M. Annu. Rev. Biophys. Bioeng. 1977, 6, 151. (41) Chalikian, T. V.; Totrov, M.; Abagyan, R.; Breslauer, K. J. J. Mol. Biol. 1996, 260, 588.

able with the HSA-dicloxacillin complexes. A possible explanation of this difference in behavior of the two systems is that premicellar association of the more hydrophobic penicillin, dicloxacillin, commences at lower drug concentrations than cloxacillin and hence the plateau region observed with the latter drug corresponds to a concentration range in which free drug molecules rather than aggregates are present in solution and there is no dehydration of the complex. 4. Conclusion We have used densimetric and acoustic techniques to measure the apparent partial specific volume and adiabatic compressibility that accompany the binding of cloxacillin and dicloxacillin penicillins to human serum albumin. Our volume and compressibility data have shown that the adsorption process can be divided into two distinct steps for both penicillins: an initial pronounced increase of the volume and compressibility of the drug-protein complex with increase of penicillin concentration at low drug concentrations that is a consequence of the development of a diffuse double layer around the complex, and a subsequent decrease at higher drug concentrations because of conformational changes in the protein attributable to a more compacted tertiary structure, and a possible onset of premicellar aggregation causing dehydration of the complex because of competition for available free water. These results have been supported by microcalorimetric data. Acknowledgment. The project was supported by the Ministerio de Ciencia y Tecnologı´a through project MAT2001-2877 and Xunta de Galicia. P. T. thanks the Ministerio de Educacion y Cultura for his Ramo´n y Cajal position. LA035106X