Interaction between Penicillins and Human Serum Albumin: A ζ

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Interaction between Penicillins and Human Serum Albumin: A ζ-Potential Study Pablo Taboada,† Victor Mosquera,† Juan M. Ruso,† Felix Sarmiento,† and Malcolm N. Jones*,‡ Grupo de Fisica de Coloides y Polymeros, Departamento de Fisica Aplicada y Departamento de Fisica de la Materia Condensada, Facultad de Fisica, Universidad de Santiago de Compostela, E15706, Santiago de Compostela, Spain, and School of Biological Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom Received September 30, 1999. In Final Form: April 18, 2000 ζ-Potential measurements have been used in studying the interaction of a range of penicillins, nafcillin, cloxacillin, dicloxacillin, and flucloxacillin with human serum albumin (HSA) in aqueous solution at pH 7.4 at 25 °C. The ζ-potentials of HSA became more negative as the negatively charged penicillins bind to it. The variations of ζ-potential with drug concentration have been interpreted in terms of the theory of Ottewill and Watanabe (OW) (Kolloid-Z. 1960, 170, 132). The theory has been modified to take into account the cooperativity of drug binding in terms of the Hill equation. The Hill coefficients for the drugs are all greater than unity and diagnostic of positive cooperativity on binding. By application of both the OW theory and its modified form, Gibbs energies per drug molecule bound (∆Gνj) as a function of the number of drug molecules bound per protein molecule were obtained. These plots are of the form and magnitude similar to those obtained from direct binding measurement, using the equilibrium dialysis technique. The application of the Hill equation to adsorption is consistent with clustering of drug molecules in micelle-like aggregates to the polypeptide chain of the protein.

Introduction In a previous study the thermodynamics of the interaction of a range of synthetic penicillins with human serum albumin (HSA) have been investigated.1 The penicillins, nafcillin, cloxacillin, dicloxacillin, and flucloxacillin were found to bind extensively to HSA in aqueous solution. The binding is exothermic and dominated by large increases in entropy, characteristics that are similar to the interactions of anionic surfactants with globular proteins.2 This observation is consistent with the formation of small micelles by these drugs in aqueous solutions.3,4 The interest in the interactions of penicillins with HSA arises from reports of allergic reactions in some subjects to penicillin and other β-lactam antibiotics5-8 and to evidence of haptenation of penicillin by conjugation with serum proteins,9-12 resulting in the formation of IgE antibodies.12-14 Penicillin G conjugates to HSA in vitro to * To whom correspondence should be addressed. E-mail: mjones@ fs1.scg.man.ac.uk. † Universidad de Santiago de Compostela. ‡ University of Manchester. (1) Taboada, P.; Mosquera, V.; Ruso, J. M.; Sarmiento, F.; Jones, M. N.; Langmuir 2000, 16, 934. (2) Jones, M. N. In Surface Activitiy Of Proteins; Magdassi, S., Ed.; Marcel Dekker: New York, 1996; Chapter 9, pp 269. (3) Taboada, P.; Attwood, D.; Ruso, J. M.; Sarmiento, F.; Mosquera, V. Langmuir 1999, 15, 2022. (4) Taboada, P.; Attwood, D.; Garcia, M.; Ruso, J. M.; Sarmiento, F.; Mosquera, V. Langmuir 2000, 16, 3175. (5) Sullivan, T. J. Pediatr. Infect. Dis. 1982, 1, 344. (6) Wen, Z. M.; Ye, S. T. Asian Pacific J. Allergy Immunol. 1993, 11, 13. (7) Audicana, M.; Bernaola, G.; Urrutia, I.; Echechipia, S.; Gastaminza, G.; Munoz, D.; Fernandez, E.; Decores, L. F. Allergy 1994, 49, 108. (8) Brander, C.; Maurihellweg, D.; Bettens, F.; Rolli, H.; Goldman, M.; Pilcher, W. J. J. Immunol. 1995, 155, 2670. (9) DiPiro, J.; Adkinson, N. F.; Hamilton, R. G. Antimicrob. Agents Chemother. 1993, 37, 1461. (10) LaFaye, P.; Lapresle, C. Febs Lett. 1987, 220, 206. (11) LaFaye, P.; Lapresle, C. Febs Lett. 1988, 234, 305. (12) Lafaye, P.; Lapresle, C. J. Clin. Invest. 1988, 82, 7.

the extent of 3.2 mol/mol of HSA but additional noncovalent binding also occurs.15 The interaction of penicillins with HSA arises both from the hydrophobic nature of the drugs3,4 and the presence of hydrophobic cavities in the protein structure.16 In this study the interaction of anionic penicillins with HSA has been investigated by measurements of the ζ-potentials of the complexes formed. Previous studies have reported investigations of the interactions of alkyl sulfates,17,18 alkyltrimethylammonium bromides,17,19 and poly(ethylene glycol)s17 with lysozyme using ζ-potential measurements, which have been interpreted in terms of the theory of Ottewill and Watanabe.20 This theory describes the adsorption of counterions to colloidal particles by the Langmuir isotherm, applicable to independent and identical adsorption sites. In the case of amphipathic counterions (e.g., penicillins) the adsorption isotherms display a degree of cooperativity on binding.1 To take cooperativity into account the Ottewill-Watanabe (OW) theory has been modified by using a Hill adsorption isotherm.21 The ζ-potential data have been interpreted in terms of the modified theory and the results used to calculate the Gibbs energies of binding. The Gibbs energies of drug binding have been compared with those obtained from the Ottewill-Watanabe theory based on a Langmuir (13) Blanca, M.; Vega, J. M.; Garaa, J.; Sanchez, F.; Perezestrada, M.; Carmona, M. J. J. Allergy Clin. Immunol. 1992, 89, 299. (14) Blanca, M.; Mayorga, C.; Perez, F.; Suau, R.; Juarez, C.; Vega, J. M.; Carmona, M. J.; Perezestrada, M.; Garcia, J. J. Immunol. Methods 1992, 153, 99. (15) Kuipers, P. J.; Fhueson, D. O.; Conroy, M. C.; Wright, C. D. FASEB J. 1988, 2, A1112. (16) He, X. M.; Carter, D. C. Nature 1992, 358, 209. (17) Kayes, J. B. J. Colloid Interface Sci. 1976, 56, 426. (18) Sarmiento, F.; Ruso, J. M.; Prieto, G.; Mosquera, V. Langmuir 1998, 14, 5725. (19) Mosquera, V.; Ruso, J. M.; Prieto, G.; Sarmiento, F. J. Phys. Chem. 1996, 100, 16749. (20) Ottewill, R. H.; Watanabe, A. Kolloid-Z. 1960, 170, 132. (21) Hill, A. V. Biochem. J. 1914, 1, 471.

10.1021/la9912904 CCC: $19.00 © 2000 American Chemical Society Published on Web 07/25/2000

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isotherm and the values obtained from equilibrium dialysis measurements. Experimental Section Materials. Human serum albumin (Type A), cloxacillin ([5methyl-3-(o-chlorophenyl-4-isoxazolyl]penicillin), sodium salt (product no. C9393), dicloxacillin ([3-(2,6-dichlorophenyl)-5methyl-4-isoxazolyl]penicillin), sodium salt monohydrate (product no. D9016), and nafcillin (6-[2-ethoxy-1-naphthamidol]penicillin), sodium salt (product no. 3269) were obtained and used as supplied from Sigma Chemical Co. Sodium flucloxacillin monohydrate([3-(2-chloro-6-fluorophenyl)-5-methyl-4-isoxazolyl]penicillin) was a generous gift from SmithKline Beecham Pharmaceuticals. Experiments were carried out, using solutions of phosphate-buffered saline (PBS, pH 7.4) prepared from PBS tablets (code BR 14a) from Oxoid, Hants, U.K. and double-distilled deionized and degassed water. ζ-Potential Measurements. The ζ-potentials of the HSA and HSA plus the drugs in PBS were measured using a Malvern Instruments Ltd, Zetasizer 3000. The ionic strength of PBS is 0.188 M so that the Debye length (1/κ) is 0.70 nm at 25 °C. Assuming a protein radius (a) of approximately 3 nm estimated from a molecular mass of 66 00022 and a partial specific volume of 0.733 cm3 g-1,23 the product κa is 4.04, corresponding to a Henry factor f(κa) of 1.14.24 The ζ-potentials (ζ)were calculated from the electrophoretic mobilities (u) from the equation

ζ)

3ηu 1 20r (1.14)

(1)

where the permittivity of vacuum (0), relative permittivity (r), and viscosity of water (η) were taken as 8.854 × 10-12 J-1 C2 m-1, 78.5, and 8.904 × 10-4 N m-2 s, respectively. For the measurements on HSA in the absence of the drugs, the required pH was obtained by the addition of hydrochloric acid or sodium hydroxide to the solutions of protein in PBS.

Equation 4 is derived from the Debye-Huckel approximation for the solution of the Poisson-Boltzmann (PB) equation since the PB equation cannot be solved analytically for any value of the potential Ψd. The DebyeHuckel approximation is only rigorously valid for small surface potentials, 1, adsorption is positively cooperative. Negative cooperativity occurs when nH < 1 and when nH ) 1 adsorption is Langmuirian. For a spherical particle of radius a, the change in diffuse layer charge (∆Q) can be related to the change in potential (∆Ψd) as follows:25

-∆σd )

(6)

It can be shown that the change in ζ-potential with concentration c is given by

where k1 ) zeN1k2 in which z is the charge on the absorbing ions, e the electronic charge, N1 the total number of adsorption sites per unit area on the adsorbing particle, and k2 the association constant. For cooperative adsorption, eq 2 may be replaced by

(k1c)nH

(k2c)nH

0r(1 + κa) 1 + (k2c)nH

A)-

In the OW theory the change in surface charge (∆σd) due to adsorption of amphipathic ions at free concentration c is given by a Langmuir expression,

-∆σd )

(zeN1)nHa

For adsorption of a negative molecule of charge z ) -1, eq 6 can be simplified by defining a parameter A given by

Theory

k1c

(5)

(10)

where

B ) (2scnH - AnHcnH-1) Solution of eq 10 gives

P)

-B ( xB2 - 4s2c2nH 2sc2nH

(11)

The association constant k2 may then be calculated from (24) Hunter, R. J. Zeta Potential in Colloid Science; Academic Press: London, 1981; Chapter 3. (25) Hunter, R. J. Zeta Potential in Colloid Science; Academic Press: London, 1981; Chapter 2, p 32. (26) Hunter, R. J. Zeta Potential in Colloid Science; Academic Press: London, 1981; Chapter 2, p 25.

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Figure 2. ζ-Potential of human serum albumin (0.125% w/v) in aqueous phosphate buffered saline (ionic strength, 0.188 M; pH 7.4; 25 °C) as a function of nafcillin concentration (9) and cloxacillin concentration (b). Figure 1. ζ-Potential of human serum albumin (0.125% w/v) in aqueous phosphate saline (ionic strength, 0.188 M) as a function of pH at 25 °C.

k2 ) P1/nH and the Gibbs energy of association per ion bound (∆Gνj) by

∆Gνj ) -RT ln 55.6k2

(12)

It should be noted that the association constant k2 is only a constant for the formation of a specific complex, i.e., HSA plus a given number of drug molecules. As binding proceeds, k2 and hence ∆Gνj change with νj due to the effect of the already bound drug molecules on further binding. The factor 55.6 converts the molar concentration to mole fraction. The values of ∆Gνj taken from equilibrium dialysis data1 (shown below) were calculated relative to a 1 M standard state; however, for the large values of νj here this makes a negligible difference. The correction factor to convert ∆Gνj to a mole fraction standard state is approximately 10/νj kJ mol-1. To calculate ∆Gνj from the ζ-potential data (Figures 2-5), values of s {)(dζ/dc)} were calculated as a function of c and used in eq 11 with the measured Hill coefficients to obtain k2 as a function of c. For a given drug concentration the binding isotherms previously reported1 were used to obtain values of νj, and hence k2 and ∆Gνj could be obtained as a function of νj. Results and Discussion Figure 1 shows the ζ-potentials of HSA as a function of pH, which were arbitrarily fitted to a fourth-order polynomial by regression analysis to give the relationship

ζ (mV) ) 67.334 - 3.216(pH) - 7.415(pH)2 + 1.281(pH)3 - 0.610(pH)4 (13) with a determination coefficient r2 ) 0.989. This relationship gives a zero ζ-potential at a pH of approximately 4.2, which compares with reported isolectric points of 4.927 and 4.7-4.9.28 The ζ-potentials of HSA in the presence of the drugs nafcillin, cloxacillin, dicloxacillin, and flucloxacillin at pH 7.4 are shown in Figures2 and 3. For nafcillin, cloxacillin, and flucloxacillin, the data cover drug con(27) Houska, M.; Brynda, E J. Colloid Interface Sci. 1997, 188, 243. (28) Bundschuh, I.; Jacklemeyer, I.; Luneberg, E.; Bentzel, C.; Petzoldt, R.; Stolte, H. Eur. J. Clin. Chem. Biochem. 1992, 30, 651.

Figure 3. ζ-Potential of human serum albumin (0.125% w/v) in aqueous phosphate-buffered saline (ionic strength, 0.188 M; pH 7.4; 25 °C) as a function of dicloxacillin concentration (b) and flucloxacillin concentration (9).

centrations below the critical micelle concentrations previously reported.3,4 Dicloxacillin (Figure 3) has two cmc’s, the first associated with spherical micelle formation (at log [dicloxacillin] ) -1.98) and the second with a spherical micelle to rodlike transition (at log [dicloxacillin] ) -1.16)3. Some of the ζ-potential data relate to total drug concentrations between the first and second cmcs, which may relate to the greater curvature in the ζ-potential-log(concentration) plots for dicloxacillin compared with those of the other drugs. The binding of the drugs to HSA previously reported,1 when analyzed using a Scatchard plot, gave parabolic curves characteristic of positive cooperativity and the formation of “micelle-like” clusters on the polypeptide chain of the protein. Regression analysis of the Scatchard plots gave the following values for the maximum number of binding sites per HSA molecule: nafcillin, 3970 ( 2190; cloxacillin, 4520 ( 2200; dicloxacillin, 1440 ( 210; flucloxacillin, 2550 ( 380. The large errors in the estimates for nafcillin and cloxacillin arise from the extrapolation of the data to maximum binding. The effect of these errors on the calculations of the Gibbs energies of binding is shown in Figures 6 and 7 below. The maximum binding values were used to draw the Hill plots shown in Figures 4 and 5. For nafcillin and cloxacillin (Figure 4) the Hill coefficients (nH) were 1.39 (r2 ) 0.932) and 1.71 (r2 ) 0.882), respectively, and for dicloxacillin and flucloxacillin (Figure

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Taboada et al. Table 1. Estimates of the Number of Binding Sites per Unit Area of Protein Surface and Areas per Drug Molecule for Drug Binding to Human Serum Albumin (HSA)

drug

N1 (drug binding sitesa m-2)

area per drug molecule (m2)

nafcillin cloxacillin dicloxacillin flucloxacillin

3.08 × 1019 3.50 × 1019 1.12 × 1019 1.98 × 1019

3.25 × 10-20 2.86 × 10-20 8.93 × 10-20 5.05 × 10-20

area per drug molecule from surface tension (m2)28 98 × 10-20 102 × 10-20 70 × 10-20

a Calculated from the total number of binding sites and the geometric area of the protein as described in the text.

described above, a radius of approximately 3 nm can be calculated from the molecular mass (M) and partial specific volume (vj 2) from the equation for the hydrodynamic volume (vh) given by29 Figure 4. Hill plots (ln(ν/(n - ν)) vs log(free drug concentration, [M]free)), where ν is the number of drug molecules bound per molecule of human serum albumin (HSA) and n is the maximum number bound for nafcillin (b) and cloxacillin (O). The data taken from ref 1 are for an HSA concentration of 0.125% w/v in aqueous phosphate-buffered saline, ionic strength of 0.188 M at 25°.

Figure 5. Hill plots (ln νj/(n - νj)) vs log(free drug concentration, [M]free)), where νj is the number of drug molecules bound per molecule of human serum albumin (HSA) and n is the maximum number bound) for dicloxacillin (O) and flucloxacillin (9). The data, taken from ref 1 are for an HSA concentration of 0.125% w/v in aqueous phosphate-buffered saline, ionic strength of 0.188 M at 25 °C.

5) the Hill coefficients were 1.23 (r2 ) 0.859) and 1.75 (r2 ) 0.883), respectively. To apply the theory described above, it is necessary to have a value for A (eq 7) as well as values of s as a function of free drug concentration c, to solve eq 10 for P and hence to obtain the binding constant k2 (P1/nH). The values of s were obtained by fitting the ζ-potential data to polynomials that were differentiated to obtain s at the required concentrations of drug. The fitting was done up to the critical concentrations of the drugs1 and in the case of dicloxacillin up to the second critical concentration.1 To calculate A, estimates of N1 (the number of binding sites per unit area of the protein surface) are required. Assuming the protein approximates to a sphere as

vh )

M (vj + δ1v01) N 2

(14)

Assuming δ1 ≈ 0.2 g of water/g of protein, v01 ≈ 1 × 10-3 m3 kg-1 and vj 2 ≈ 0.7 × 10-3 m3 kg-1. Alternatively, the volume of the HSA unit cell16 is 0.137 × 10-24 m3; if this volume corresponded to a sphere, its radius would be 3.2 nm and its surface area 1.29 × 10-16 m2. From this surface area and the total number of binding sites taken from the Scatchard plots, estimates of the number of sites per unit area were made and are given in Table 1 (column 2). The areas per drug molecule calculated from these estimates are compared with areas per drug molecule at the aqueous-air interface derived from surface tension data30 (Table 1, column 4). The areas at the aqueous-air interface are much larger than those estimated for adsorption at the protein-aqueous interface. This may well arise because the surface area of HSA, as estimated above, is essentially a “geometric area”, which will be less than the true topographic area of the protein. If the true area were known, it would give a much larger area per drug molecule. The values of N1 from the Scatchard analysis show a decrease by approximately 3 for dicloxacillin compared with that for cloxacillin, which may be attributed to the additional chloride atom in dicloxacillin. This leads to a larger area per dicloxacillin molecule in column 3 and a significant decrease in the ratio of the area per drug molecule from surface tension measurements compared to those from binding measurements (this ratio of areas, column 4:column 3, is ∼8). For the other two drugs the ratios are similar, ∼30 (nafcillin) and ∼35 (cloxacillin). HAS has 17 disulfide bridges16 that will restrict the molecule to a compact structure; the stereoview of the molecule shows it to have a pyramidal shape. While the sphere model has deficiencies, it is not clear what better model could be used. Figures 6-9 show the Gibbs energies of drug binding as a function of the number of drug molecules bound per HSA molecule calculated from equilibrium dialysis measurements1 and from the ζ-potential measurements using the above theory based on both the Langmuir and Hill adsorption isotherms. For all the drugs apart from flucloxacillin, the theory based on the Hill equation gives ∆Gνj vs νj plots closer to those obtained from direct binding measurements than does the unmodified OW theory based (29) Tanford, C. Physical Chemistry of Macromolecules; John Wiley &Sons: New York, 1967; Chapter 20, p 339. (30) Taboada, P.; Attwood, D.; Ruso, J. M.; Garcia, M.; Sarmiento, F.; Mosquera, V. J. Colloid Interface Sci. 1999, 220, 288.

Interaction of Penicillins and Human Serum Albumin

Figure 6. Gibbs energies of binding of nafcillin to human serum albumin (HSA), as a function of the number of nafcillin molecules bound per HSA molecule (νj). (b) Experimental values calculated from direct binding (equilibrium dialysis) measurements (ref 1). Values calculated from ζ-potential measurements based on a Langmuir adsorption isotherm (9) and cooperative binding (2) with a Hill coefficient nH ) 1.39. The error bars are based on the errors in the estimates of the maximum number of binding sites as indicated in the text. All the data are for aqueous phosphate-buffered saline solutions, pH 7.4 and ionic strength of 0.188 M at 25 °C.

Figure 7. Gibbs energies of binding of cloxacillin to human serum albumin (HSA), as a function of the number of cloxacillin molecules bound per HSA molecule (νj). (b) Experimental values calculated from direct binding (equilibrium dialysis) measurements (ref 1). Values calculated from ζ-potential measurements based on a Langmuir adsorption isotherm (9) and cooperative binding (2) with a Hill coefficient nH ) 1.71. The error bars are based on the errors in the estimates of the maximum number of binding sites as indicated in the text. All the data are for aqueous phosphate-buffered saline solutions, pH 7.4 and ionic strength of 0.188 M at 25 °C.

on a Langmuir adsorption isotherm. The shapes of the ∆Gνj vs νj plots derived from the ζ-potentials show initial binding to higher energy sites (more negative ∆Gνj values) followed by weaker interaction as binding proceeds. In terms of the numerical values of ∆Gνj at νj ) 500, the strength of binding follows the sequence nafcillin (-9.7 kJ mol-1) > dicloxacillin (-8.3 kJ mol-1) > cloxacillin (-7.0 kJ mol-1) > flucloxacillin (-6.0 kJ mol-1). The electrical contributions to the Gibbs energies of binding can be approximately estimated from the expression (zieξi where zi is the charge on the adsorbing ion (here, -1), e is the electronic charge per mole, and ξi the ζ-potential.31 From the ζ-potentials (Figures 2 and 3) and binding data1 the (31) Hunter, R. J. Zeta Potential in Colloid Science; Academic Press: London, 1981; Chapter 2, p 34.

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Figure 8. Gibbs energies of binding of dicloxacillin to human serum albumin (HSA), as a function of the number of dicloxacillin molecules bound per HSA molecule (νj). (b) Experimental values calculated from direct binding (equilibrium dialysis) measurements (ref 1). Values calculated from ζ-potential measurements based on a Langmuir adsorption isotherm (9) and cooperative binding (2) with a Hill coefficient nH ) 1.23. All the data are for aqueous phosphate-buffered saline solutions, pH 7.4 and ionic strength of 0.188 M at 25 °C.

Figure 9. Gibbs energies of binding of flucloxacillin to human serum albumin (HSA), as a function of the number of flucloxacillin molecules bound per HSA molecule (νj). (b) Experimental values calculated from direct binding (equilibrium dialysis) measurements (ref 1). Values calculated from ζ-potential measurements based on a Langmuir adsorption isotherm (9) and cooperative binding (2) with a Hill coefficient nH ) 1.75. All the data are for aqueous phosphate-buffered saline solutions, pH 7.4 and ionic strength of 0.188 M at 25 °C.

electrical contributions at νj ) 500 follow the sequence flucloxacillin (-4.0 kJ mol-1) > cloxacillin (-3.7 kJ mol-1) > nafcillin (-3.1kJ mol-1) > dicloxacillin (-2.5 kJ mol-1). These electrical contributions are 32% (nafcillin), 30% (dichloxacillin), 53% (cloxacillin), and 67% (flucloxacillin) of the total Gibbs energies of binding. Summary In summary, the application of ζ-potential measurements to the study of drug-protein interactions gives Gibbs energies of interactions as a function of the numbers of drug molecules bound that are of similar shape and magnitude to data obtained by direct measurement of the extents of drug binding. Except for flucloxacillin, when the OW theory is modified to take into account the cooperativity of binding by use of the Hill adsorption isotherm, the Gibbs energies of binding are closer to the

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experimental values from equilibrium dialysis experiments than when the Langmuir isotherm is used. The modified OW theory is consistent with the clustering of drug molecules on the polypeptide chain of the protein and a description of the resulting complexes in which the protein is covered in micelle-like aggregates.

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Acknowledgment. We thank the Xunta de Galicia for financial support and Fundacion Caixa Galicia for a grant for P.T. LA9912904