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Langmuir 2003, 19, 1446-1448
Protein-Ligand Interactions: Volumetric and Compressibility Characterization of the Binding of Two Anionic Penicillins to Human Serum Albumin
content and temperature. Cloxacillin and dicloxacillin are structurally similar, differing only in an additional chlorine atom on the phenyl ring of dicloxacillin (see structure). They are anionic molecules with pKa values of
Silvia Barbosa, Pablo Taboada, 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 Received September 25, 2002. In Final Form: November 18, 2002
1. Introduction 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. Human serum albumin (HSA) consists of 585 amino acids in a single polypeptide chain with a molar mass of 66 411 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 a 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.2 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 the internal cavity.3,4 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 structures5 in the neighborhood of the hydrophobic groups and (2) shrinkage due to the intrusion of the hydrophobic groups into empty spaces inherent in the water structure. The overall volume effect seems to be dominated by the latter, that is, filling of clathrate cages, in most biochemical processes.6 It is expected that in the protein-penicillin complex formation some possible structural deformation may also occur as a result of the interaction between the anionic protein molecule and the anionic amphiphile penicillin molecule. Our previous studies of the solution properties of a large number of penicillins7-13 have characterized the selfassembly in aqueous solution as a function of electrolyte * To whom correspondence should be addressed. E-mail:
[email protected]. Telephone: 0034981563100 Ext 14056. Fax: 0034981520676. (1) Peters, T. J. All about Albumin Biochemistry, Genetics, and Medical Applications; Academic Press: San Diego, CA, 1996. (2) Richards, F. M. Annu. Rev. Biophys. Bioeng. 1977, 6, 151. (3) Scharade, P.; Klein, H.; Egry, I.; Ademovic, Z.; Klee, D. J. Colloid Interface Sci. 2001, 234, 445. (4) Nemethy, G.; Scheraga, H. A. J. Chem. Phys. 1962, 36, 3401. (5) Neal, J. L.; Goring, D. A. J. Phys. Chem. 1970, 74, 658. (6) Iqbal, M.; Verral, R. E. J. Phys. Chem. 1987, 91, 1935. (7) Taboada, P.; Attwood, D.; Ruso, J. M.; Sarmiento, F.; Mosquera, V. Langmuir 1999, 15, 2022. (8) Taboada, P.; Attwood, D.; Ruso, J. M.; Garcı´a, M.; Sarmiento, F.; Mosquera, V. Langmuir 2000, 16, 3175.
2.7 and 2.8, respectively, and will be fully ionized in aqueous solution. Static light scattering and NMR studies7 have shown that both drugs form small aggregates (typically five to six molecules) at a well-defined critical micellar concentration (cmc) in aqueous solution. In a previous work13 we have compared the adsorption characteristics of the two structurally similar penicillin drugs on the surface of human serum albumin. With both drugs the adsorption was accompanied by a gradual change in hydrodynamic radius of the complex with increasing drug concentration indicative of saturation rather than a denaturation process, with saturation of the protein surface, at a fixed HSA concentration of 6.25 × 10-4 g cm-3, occurring at drug concentrations of approximately 0.064 and 0.056 g cm-3 for cloxacillin and dicloxacillin, respectively. The number of available adsorption sites per unit area of protein was appreciably larger for the cloxacillin monomer, a larger number of which were adsorbed (approximately 2500 compared to 1050 for dicloxacillin), with the difference reflecting the larger area occupied on the protein surface by the dicloxacillin molecules as a consequence of the additional Cl atom. Similarly, a larger free energy change (per molecule) on adsorption for the more hydrophobic dicloxacillin amphiphile molecule was observed. HSA/dicloxacillin complexes were of larger size, suggesting a more appreciable extension or unfolding of the HSA molecule than in the HSA/cloxacillin systems. 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 changes.14 It is known that the adiabatic compressibility sensitively reflects the characteristic structures of native proteins15,16 and the conformational changes induced by denaturation17 although it is a macroscopic quantity involving two contributions, surface hydration and internal cavity due to imperfect atomic packing. In the case of an amphiphilic molecule dissolved in water, (9) Taboada, P.; Attwood, D.; Garcı´a, M.; Jones, M. N.; Ruso, J. M.; Mosquera, V.; Sarmiento, F. J. Colloid Interface Sci. 2000, 221, 242. (10) Taboada, P.; Attwood, D.; Ruso, J. M.; Garcı´a, M.; Sarmiento, F.; Mosquera, V. J. Colloid Interface Sci. 1999, 216, 270. (11) 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. (12) Taboada, P.; Attwood, D.; Ruso, J. M.; Garcı´a, M.; Sarmiento, F.; Mosquera, V. J. Colloid Interface Sci. 1999, 220, 288. (13) Ruso, J. M.; Attwood, D.; Garcı´a, M.; Taboada, P.; Varela, L. M.; Mosquera, V. Langmuir 2001, 17, 5189. (14) Cooper, A. Thermodynamic fluctuations in protein molecules. Proc. Natl. Acad. Sci. U.S.A. 1976. (15) Gekko, K.; Noguchi, H. J. Phys. Chem. 1979, 83, 2706. (16) Chalikian, T. V.; Totrov, M.; Abagyan, R.; Breslauer, K. J. J. Mol. Biol. 1996, 260, 588. (17) Kharakoz, D. P. Biochemistry 1997, 36, 10276.
10.1021/la020810j CCC: $25.00 © 2003 American Chemical Society Published on Web 01/22/2003
Notes
Langmuir, Vol. 19, No. 4, 2003 1447
the water reorganization gives as a result a negative contribution to the total 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 must be reflected in thermodynamic properties related to the volume and compressibility of the implicated species. The compressibility changes of proteins due to ligand binding should shed light on how conformational flexibility manifests its effect through the changes in internal cavities and solvent accessible surface area. In the present work, we report the results of our studies of 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, as a function of the concentration of the penicillin drugs cloxacillin and dicloxacillin. For such a globular protein it has been previously shown by Gekko et al.18,19 that the protein concentration dependences of v and βS are negligible at concentrations between 0 and 5 g dm-3. The data reported here are for low concentrations and, for the amphiphilic substances, below the critical micellar concentration, so their behavior could be considered to be that of a 1:1 electrolyte. This treatment has been widely used to obtain interactions between proteins and surfactants, but to our knowledge it has not previously been applied to systems of the type considered here. 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 using double distilled, deionized, and degassed water. 2.2. Adsorption of Cloxacillin and Dicloxacillin onto Albumin. To prepare the dissolutions, aliquots of 2.5 cm3 of a 0.4% (w/v) solution of HSA were added to equal volumes of penicillin aqueous solutions to give a final solution in which the concentration of HSA was 0.2% (w/v). The concentrations of both penicillins were kept always below the first critical concentration (0.065 and 0.058 g cm-3 for cloxacillin and dicloxacillin and in the presence of a 0.0625% (w/v) solution of HSA, respectively). 2.3. Density and Ultrasound Velocity Measurements. Density and ultrasound velocity measurements were carried out using a commercial density and ultrasound velocity measurement apparatus (Anton Paar DSA 5000 densimeter and sound velocity analyzer). The temperature control was maintained within (0.001 K by the Peltier method, giving rise to uncertainties in density of ∼ (1 × 10-6 g cm-3. Errors in ultrasound velocity measurements arise mainly from variations of temperature, and in this study the accuracy in the velocity was (0.01 m s-1. The experimental procedures were essentially the same as those used in the previous study.20 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 a sound velocity, u, and density, F, data set of the sample solutions and solvents (u0 and F0) at the temperature 30 °C and different penicillin concentrations always below the critical micelar concentration,
v)
βS ) -
[
]
1 F-c 1c F0
( ) ( )[
1 ∂v v ∂p
)
S
(1)
]
β0 β F-c vc β0 F0
(2)
(18) Gekko, K.; Hasegawa, Y. Biochemistry 1986, 25, 6563. (19) Gekko, K.; Hasegawa, Y. J. Phys. Chem. 1989, 93, 426. (20) Gutie´rrez-Pichel, M.; Taboada, P.; Varela, L. M.; Attwood, D.; Mosquera, V. Langmuir 2002, 18, 3650.
where p is the pressure, c is the concentration of the solute (protein in g cm-3), and β and β0 are the adiabatic compressibilities of sample solution and solvent, respectively, which were calculated with the Laplace equation, β ) 1/(Fu2).
3. Results and Discussion The apparent partial specific volume, v, and adiabatic compressibility, βS, of HSA and its ligand complexes are macroscopic observables 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.21,22 The HSA-penicillin complex formation may be thought of as proceeding in three states:23 (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. 3.1. Volume Changes. The apparent partial specific volume of a protein in water, v, can be considered to be the sum of the following terms:24
v ) va + vc + ∆vh + βT0RT
(3)
where va is the constitutive atomic volume, vc is the internal cavity due to imperfect atomic packing, and ∆vh is the volume change due to hydration that 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 produces a negative volume change; then ∆vh is ordinarily 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, βT0RT, describes the volume effect related to the kinetic contribution to the pressure of a solute molecule due to the translational degrees of freedom.25 The value of this term is about 1 cm3 mol-1 and usually can be ignored when considering large solutes such as proteins. Figure 1 shows the partial specific volume of HSApenicillin complexes as a function of penicillin concentration. The adsorption process suggested by these data can be divided into two distinct steps for both penicillins. There is an initial pronounced increase of the protein volume up to a penicillin concentration of approximately 0.01 g cm-3, as a consequence of increasing adsorption of the negative charged penicillin monomer as the drug concentration is increased. As can be observed in the figure, the volumes of HSA-ligand are practically the same for both penicillins. The only possible specific protein-ligand interaction is of a hydrophobic type. In general, the solute-solute interactions in water accompany dehydration of each solute molecule, resulting in the increase in volume. The protein volume versus drug concentration at penicillin concentrations greater than approximately 0.01 g cm-3 decreases with penicillin concentration, with the volume (21) Chalikian, T. V.; Totrov, M.; Abagyan, R.; Breslauer, K. J. J. Mol. Biol. 1996, 260, 588. (22) Chalikian, T. V.; Sarvazyan, A. P.; Breslauer, K. J. Byophys. Chem. 1994, 51, 89. (23) Kamiyama, T.; Gekko, K. Biochim. Biophys. Acta 2000, 1478, 257. (24) Dubins, D. N.; Filfil, R.; Macgregor, R. B.; Chalikian, T. V. J. Phys. Chem. B 2000, 104, 390. (25) Stillinger, F. H. J. Solution Chem. 1973, 2, 141.
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Langmuir, Vol. 19, No. 4, 2003
Notes
Figure 1. Apparent partial specific volume, v, of HSA as a function of concentration, c, at 30 °C: cloxacillin (9); dicloxacillin (4).
Figure 2. Apparent partial specific adiabatic compressibility, βS, of HSA as a function of concentration, c, at 30 °C: cloxacillin (9); dicloxacillin (4).
reduction in the case of dicloxacillin being more pronounced. This implies, for both penicillins, that the dehydration effects would be totally overcome by atomic packing effects due to conformational changes in the protein. This result, which will be confirmed by compressibility data, suggests that the tertiary structure becomes more compact upon binding. 3.2. Compressibility Changes. Since the constitutive atomic volume may be assumed as incompressible, the apparent partial specific adiabatic compressibility of a protein, βS, is mainly determined by the internal cavity and surface hydration as follows:15,18
( )( ) ( )(
βS ) -
1 ∂v v ∂p
)-
S
)
1 ∂vc ∂(∆vh) + v ∂p ∂p
S
) βc + βh
(4)
where βS ) -(1/v)(∂∆vc/∂p)S is the intrinsic compressibility of the protein molecule and reflects the imperfect packing of the polypeptide chain(s). For a globular protein, βc contributes positively to the adiabatic compressibility of the protein.23 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 water molecules.26 For a globular protein, βc contributes negatively to the adiabatic compressibility of the protein. Figure 2 shows the apparent partial specific adiabatic compressibility of HSA-penicillin complexes as a function of penicillin concentration. The adsorption process suggested by the compressibility data reflects the same behavior as the volumetric data. The adsorption process can be divided into two distinct steps for both penicillins, with an initial pronounced increase of the protein compressibility up to a penicillin concentration of approximately 0.01 g cm-3 that reflects the dehydration of each solute molecule and a posterior decrease due to conformational changes in the protein. Figure 3 shows plots of βS against v for the HSA-ligand complexes. The correlations of the least-squares linear regressions are 0.9965 and 0.972 for cloxacillin and dicloxacillin, respectively. The figure shows that βS increases with increasing v, as found statically for other (26) Chalikian, T. V.; Totrov, M.; Abagyan, R.; Breslauer, K. J. J. Mol. Biol. 1996, 260, 588.
Figure 3. Plots of βS against v for HSA-ligand complexes: cloxacillin (9); dicloxacillin (4).
protein systems.23 This is a clear evidence that the internal atomic packing contributes positively and the surface hydration negatively to v and βS. 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. We describe how the changes in our volumetric and compressibility data can be ascribed to the proteinligand complex formation. Our volume and compressibility data suggest the adsorption process can be divided into two distinct steps for both penicillins, with an initial pronounced increase of the protein volume and compressibility with penicillin concentration that reflects the dehydration of each solute molecule and a posterior decrease due to conformational changes in the protein that can be attributed to a more compacted tertiary structure. 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. LA020810J
