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Langmuir 2000, 16, 934-938
Interaction between Penicillins and Human Serum Albumin: A Thermodynamic Study of Micellar-like Clusters on a Protein Pablo Taboada, Victor Mosquera, Juan M. Ruso, and Felix Sarmiento Grupo de Fisica de Coloides y Polimeros, Departamento de Fisica Aplicada y Departamento de Fisica de la Materia Condensada, Facultad de Fisica, Universidad de Santiago de Compostela, E-15706 Santiago de Compostela, Spain
Malcolm N. Jones* School of Biological Sciences, University of Manchester, Manchester M13 9PT, U.K. Received May 4, 1999. In Final Form: August 16, 1999 The thermodynamic parameters for the interaction of a range of penicillins, nafcillin, cloxacillin, dicloxacillin, and flucloxacillin, with human serum albumin (HSA) in aqueous solution, pH 7.4, 25 °C, have been determined using a combination of equilibrium dialysis and microcalorimetric techniques. The drugs bind largely nonspecifically to HSA to various extents ranging from ∼1200 (dicloxacillin) to ∼3000 (nafcillin) drug molecules per HSA molecule as the free drug concentration approaches the critical concentration (cc) for aggregation of the free drug. Maxima in the binding isotherms are found for nafcillin and cloxacillin, which possibly relate to maxima in monomeric drug activity in the vicinity of the critical concentrations (ccs). In the case of the dicloxacillin-HSA system, the binding isotherm reflects the two ccs observed for dicloxacillin corresponding to aggregate formation and the sphere-to-rod aggregate transition. The enthalpies of binding are small and exothermic so that the Gibbs energies of binding are dominated by large increases in entropy consistent with hydrophobic interactions. The magnitudes of the thermodynamic parameters for the interactions are similar to these for the interactions of anionic surfactants with globular proteins. The results are consistent with the clustering of drug molecules to the protein to form micellar-like structures.
Introduction The chemical use of penicillin goes back over 60 years following its discovery by Fleming in 1928,1 and work on the colloidal properties of penicillin followed in the 1940s2 and 1950s.3,4 More recently the micellar properties of synthetic penicillins (cloxacillin, dicloxacillin, and flucloxacillin) have been studied.5,6 The clinical use of penicillins and other β-lactam antibiotics has been complicated by the presentation of hypersensitivity and allergic reactions in a proportion of patients.7-10 Evidence has been presented for the haptenation of penicillin by conjugation with serum proteins,11-14 which results in the * To whom correspondence should be addressed. Email:
[email protected]. (1) Stryer, L. Biochemistry, 4th ed.; W. H. Freeman: New York, 1995; p 201. (2) McBain, J. W.; Huff, H.; Bady, A. P. J. Am. Chem. Soc. 1949, 71, 373. (3) Hauser, E. A.; Marlow, G. J. J. Phys. Colloid Chem. 1950, 54, 1077. (4) Few, A. V.; Shulman, J. H. Biochim. Biophys. Acta 1953, 10, 302. (5) Attwood, D.; Agarwal, S. P. J. Pharm. Pharmacol. 1984, 36, 563. (6) Taboada, P.; Attwood, D.; Ruso, J. M.; Sarmiento, F.; Mosquera, V. Langmuir 1999, 15, 2022. (7) Sullivan, T. J. Pediatr. Infect. Dis. 1982, 1, 344. (8) Wen, Z. M.; Ye, S. T. Asian Pacific J. Allergy Immunol. 1993, 11, 13. (9) Audicana, M.; Bernaola, G.; Urrutia, I.; Echechipia, S.; Gastaminza, G.; Munoz, D.; Fernandez, E.; Decorres, L. F. Allergy 1994, 49, 108. (10) Brander, C.; Maurihellweg, D.; Bettens, F.; Rolli, H.; Goldman, M.; Pilcher, W. J. J. Immunol. 1995, 155, 2670. (11) DiPiro, J.; Adkinson, N. F.; Hamilton, R. G. Antimicrob. Agents Chemother. 1993, 37, 1463. (12) LaFaye, P.; Lapresle, C. FEBS. Lett. 1987, 220, 206. (13) LaFaye, P.; Lapresle, C. FEBS. Lett. 1988, 234, 305. (14) LaFaye, P.; Lapresle, C. J. Clin. Invest. 1988, 82, 7.
formation of IgE antibodies.14-16 The hapentenation of serum proteins is believed to be facilitated by a low molecular mass factor in serum.11 In the absence of this serum factor, the extent of hapentation of pure human serum albumin (HSA) was markedly reduced.11 The extent of conjugation of penicillin G with HSA in vitro has been reported to be 3.2 mol of penicillin per mole of HSA, but noncovalent binding also occurs.17 The hydrophobic nature of penicillins as demonstrated by their self-association behavior5,6 and the evidence of hydrophobic cavities in the structure of HSA18 suggest that noncovalent binding of penicillins is an important property of the protein requiring more detailed study. In this work the binding of a range of synthetic penicillins, nafcillin, cloxacillin, dicloxacillin, and flucloxacillin, whose structure are shown in Scheme 1, has been measured by equilibrium dialysis to obtain the binding constants and Gibbs energies of binding. These data have been used in combination with the enthalpies of interaction measured by microcalorimetry to obtain a thermodynamic picture of the nature of the interactions between the penicillins and HSA. We consider the interactions between the drug molecules and the protein to be an example of binding described by multiple equilibria. The binding process is largely non(15) Blanca, M.; Vega, J. M.; Garcia, J.; Sanchez, F.; Perezestrada, M.; Carmona, M. J. J. Allergy Clin. Immunol. 1992, 89, 299. (16) Blanca, M.; Mayorga, C.; Perez, E.; Suau, R.; Juarez, C.; Vega, J. M.; Carmona, M. J.; Perezestrada, M.; Garcia, J. J. Immunol. Methods 1992, 153, 99. (17) Kuipers, P. J.; Thueson, D. O.; Conroy, M. C.; Wright, C. D. FASEB J. 1988, 2, A1112. (18) He, X. M.; Carter, D. C. Nature 1992, 358, 209.
10.1021/la990538m CCC: $19.00 © 2000 American Chemical Society Published on Web 12/03/1999
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Scheme 1
Figure 2. Binding isotherm for cloxacillin on human serum albumin in aqueous solution, pH 7.4, 25 °C. The cc of cloxacillin is indicated on the x-axis. νj is the number of cloxacillin molecules bound per protein molecule.
Figure 1. Binding isotherm for nafcillin on human serum albumin in aqueous solution, pH 7.4, 25 °C. The cc of nafcillin is indicated on the x-axis. νj is the number of nafcillin molecules bound per protein molecule.
specific and, as we show, results in clustering of drug molecules around the protein to form micellar-like species.
Figure 3. Binding isotherm for dicloxacillin on human serum albumin in aqueous solution, pH 7.4, 25 °C. The first and second cc of dicloxacillin are indicated on the x-axis. νj is the number of dicloxacillin molecules bound per protein molecule.
Experimental Section Materials. Human serum albumin (type A) cloxacillin ([5methyl-3-(o-chlorophenyl)-4-isoxazolyl]penicillin), sodium salt monohydrate (product no. C9393), dicloxacillin ([3-(2,6-dichlorophenyl)-5-methyl-4-isoxazolyl])penicillin, sodium salt monohydrate (product no. D9016), and nafcillin (6-[2-ethoxy-1naphthamidol]penicillin, sodium salt (product no. N 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. Dialysis tubing was Spectrapor (No. 3, molecular weight cutoff 3500) from Spectrum Medical Industries, LA. Drug-Binding Measurements. Aliquots (2 cm3) of HSA (concentration 0.125% w/v in PBS, pH 7.4) in dialysis bags were equilibrated with 5 cm3 of drug solutions in a buffer over the concentration range 0-0.1 M in screw cap sample tubes held in a thermostated bath at 25 °C for a period of at least 96 h. It has been previously demonstrated that low molecular weight ligands equilibrate across Spectrapor (No. 3) in this time period.19 At equilibrium the concentrations of the free drugs were measured with reference to their extinction coefficients, which were found to be as follows: E280nm (nafcillin, Mr ) 454.5) ) 4091 ( 64 M-1 cm-1; E274nm (cloxacillin, Mr ) 475.9) ) 259 ( 5; E274nm (19) Prieto, G.; del Rio, J. M.; Paz Andrade, M. I.; Sarmiento, F.; Jones, M. N. Int. J. Biol. Macromol. 1993, 15, 343.
(dicloxacillin, Mr ) 510.3) ) 543 ( 7; E274nm (flucloxacillin, Mr ) 493.9) ) 552 ( 14. The molecular mass of HSA was taken as 66 000 g mol-1.20 Microcalorimetry. Enthalpy measurements were made at 298.15 K using an LKB-Producter 10700 twin-cell batch microcalorimetry system.21 On the most sensitive range used for the measurements (30 µV), the mean sensitivity of the detectors in the heat sinks of the two vessels was (14.66 + 0.32) µW µV-1. The two detector sensitivities differed by only 0.37%. The sample cell was charged with 2 g of buffered HSA solution, concentration 0.25% w/v and 2 g of drug solution of the required concentration. The reference cell was charged with 2 g of buffer solution and 2 g of drug solution of identical concentration to that in the sample cell. On mixing, the enthalpies of dilution of the drug solutions cancel, and the enthalpy of dilution of the HSA solution was negligible. The lower limit of signal detection is 1-2 mJ; this energy arising from frictional effects on mixing; hence, for 2 g of a 0.25% w/v protein solution, i.e., 75.8 nmol of protein, this limit corresponds to an enthalpy of approximately 13-26 kJ mol-1. If the heat of dilution of protein falls below this value, it will not be measurable under these conditions. Similarly the errors arising from variability in frictional enthalpy effects put an uncertainty of the same order on the enthalpy measurements. The scatter in the data points in Figures 5-7 reflects this error. (20) Frank, M.; Mears, C. A.; Labov, S. E.; Benner, W. H.; Hom, D.; Jaklevic, J. M.; Barknecht, A. T. Rapid Commun. Mass Spectrom. 1996, 10, 1946. (21) Wadso¨, I. Acta Chem. Scand. 1968, 22, 92.
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Figure 4. Binding isotherm for flucloxacillin on human serum albumin in aqueous solution, pH 7.4, 25 °C. The cc of flucloxacillin is indicated on the x-axis. νj is the number of flucloxacillin molecules bound per protein molecule.
Figure 5. Enthalpy of interaction of nafcillin with human serum albumin in aqueous solution, pH 7.4, 25 °C, as a function of the final drug concentration after mixing. The arrow denotes the cc.
Figure 6. Enthalpy of interaction of cloxacillin with human serum albumin in aqueous solution, pH 7.4, 25 °C, as a function of the final drug concentration after mixing. The arrow denotes the cc.
Results and Discussion The adsorption isotherms for the four drugs on human serum albumin (HSA) plotted as the number of drug molecules bound per HSA molecule (νj) as a function of the log(free drug concentration (M)) are shown in Figures 1-4.
Taboada et al.
Figure 7. Enthalpy of interaction of dicloxacillin with human serum albumin in aqueous solution, pH 7.4, 25 °C, as a function of the final drug concentration after mixing. The arrow denotes the cc.
For nafcillin and cloxacillin the isotherms pass through maxima at free drug concentrations below the critical concentrations (ccs). The critical concentrations were taken from previous studies22 and are indicated by arrows in the figures. Maxima in adsorption isotherms of surfactant binding to globular proteins23 and insoluble polymeric substrates24,25 have been previously observed and have been attributed to the activity of long-chain ions going through a maximum in the vicinity of the cmc.26,27 Dicloxacillin has been found to have two ccs, the first attributed to spherical aggregate formation and the second to a sphere-to-rod transition.22 The binding isotherm shown in Figure 3 appears to reflect these properties in that a slight plateau in νj occurs above the first cc and a steep rise in νj occurs before the second cc. The binding isotherms for the drugs range up to νj values of 3000 for nafcillin and cloxacillin, 1200 for dicloxacillin, and 2000 for flucloxacillin. Human serum albumin consists of 585 amino acids18 so binding of 3000 drug molecules corresponds to approximately 5 drug molecules per amino acid residue; this suggests clustering of drug molecules along the polypeptide chain. Such a cluster size corresponds closely to the aggregation numbers of the drug micelles, which at an ionic strength of 0.1 M were found to be 5 (cloxacillin), 5 (dicloxacillin), and 4 (flucloxacillin).22 However, these data do not enable us to draw any conclusions about the uniformity of binding along the polypeptide chain, although it is likely that binding is greater (and cluster size larger) around hydrophobic amino acid residues and in hydrophobic cavities in the protein18 and less around hydrophilic residues. The enthalpies of interaction of nafcillin, cloxacillin, and dicloxacillin with HSA are shown in Figures 5-7. Owing to the limited availability of flucloxacillin, it was not possible to make enthalpy measurements on this drug. The enthalpy plotted in these figures is per mole of HSA. The enthalpies are exothermic, are approximately linear in drug concentration at low concentrations, and approach limiting values as the drug concentrations approach the (22) Taboada, P.; Attwood, D.; Garcia, M.; Ruso, J. M.; Sarmiento, F.; Mosquera, V. Submitted for publication in Langmuir. (23) Jones, M. N.; Manley, P.; Midgley, P. J. W. J. Colloid Interface Sci. 1981, 82, 257. (24) Hall, D. G. J. Chem. Soc., Faraday Trans. 1 1980, 76, 386. (25) Sexsmith, F. H.; White, H. J. J. Colloid Sci. 1959, 14, 630. (26) Murray, R. C.; Hartley, G. S. Trans. Faraday Soc. 1935, 31, 183. (27) Mysels, K. J. J. Colloid Sci. 1955, 10, 507.
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Langmuir, Vol. 16, No. 3, 2000 937
Figure 8. Enthalpy of interaction per mole of drug (∆Hν) as a function of extent of binding to human serum albumin (νj), pH 7.4, 25 °C: (2), nafcillin; (b), cloxacillin; (9), dicloxacillin.
ccs. The data from the binding isotherms was used to produce plots of binding as a function of total drug concentration, which were then used to plot the enthalpies of interaction per mole of drug bound (∆Hν) as a function of binding (νj) (Figure 8). The data in Figure 8 show that the enthalpies per mole of drug bound become less exothermic as νj increases. Thus initial binding is to highenergy sites. For nafcillin and cloxacillin, the enthalpies of binding reach limiting values of approximately -0.04 kJ mol-1 of drug bound. Binding of dicloxacillin is more exothermic and, while initially becoming less exothermic when the high-energy sites are saturated, becomes more exothermic again as the second cc is approached. The origin of this effect is unclear but may possibly arise from some structural rearrangement of the bound drug molecules. Structurally the only difference between cloxacillin and dicloxacillin is an additional chlorine atom on the aromatic ring (see Scheme 1). This results in a substantial increase in the enthalpy of interactions as well as inducing a second cc. The Gibbs energies of binding per mole of drug (∆Gν) were calculated from the Wyman binding potential (Π) derived from the area under the binding isotherms as previously described.28
Π ) RT
∫0νjd ln[S]
(1)
where R is the gas constant, T the absolute temperature, and [S] the free drug concentration. In the cases of nafcillin and cloxacillin, the integration was only carried out up to values of ν below the maxima in the binding isotherms. The binding potential is related to the apparent binding constant (Kapp) by the equation28
Figure 9. Gibbs energy of interaction per mole of cloxacillin (∆Gν) as a function of extent of binding (νj) to human serum albumin, pH 7.4, 25 °C.
Figure 10. Thermodynamic parameters for the binding of nafcillin to human serum albumin, pH 7.4, 25 °C, as a function of extent of binding (νj).
complexes formed when large numbers of ligands are bound. Figure 9 shows ∆Gν vs νj for the cloxacillin-HSA system. ∆Gν is large and negative at low values of νj where binding to the “high-energy” sites occurs and become less negative as more drug molecules bind. Similar data were obtained for the HSA with the other drugs. The data for ∆Hν vs νj and ∆Gν vs νj were fitted to appropriate equations and used to calculate the corresponding entropy per mole of drug bound (∆Sν) shown in Figures 10-12 using the relationship
∆Gνj ) ∆Hνj - T∆Sνj
(4)
This analysis of the data takes into account the variation of the binding constants with the extent of binding that arises from multiple equilibria involving a range of
These data show that for all the drugs, binding is characterized by large increases in entropy (right-hand axes). The enthalpy (∆Hν) makes very little contribution to ∆Gν. The large entropy increases are characteristic of hydrophobic interactions between the drugs and the protein. The magnitudes of both ∆Gν and T∆Sν are very similar to those reported for the interaction of the anionic surfactant, sodium n-dodecylsulfate (SDS), with globular proteins.28 In particular the binding of the first approximately 100 SDS molecules to bovine serum albumin (BSA)29 at pH 7 and 25 °C occurs with a ∆Gν of -24.8 kJ mol-1 and a T∆Sν of +17.9 kJ mol-1. The interaction of
(28) Jones, M. N., Chapman, D. Micelles, Monolayers and Biomembranes; Wiley-Liss: New York, 1995; Chapter 6, p 161.
(29) Jones, M. N. In Surface activity of proteins; Magdassi, S., Ed.; Marcel Dekker: New York, 1996; Chapter 9, p 269.
νj
Γ ) RT ln(1 + Kapp [S] )
(2)
The Gibbs energy of binding is calculated from
∆Gνj ) -
RT ln Kapp νj
(3)
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Langmuir, Vol. 16, No. 3, 2000
Taboada et al. Table 1. Thermodynamic Parameters for the Interaction of Penicillins with Human Serum Albumin in Aqueous Solution, pH 7.4, 25 °C
Figure 11. Thermodynamic parameters for the binding of cloxacillin to human serum albumin, pH 7.4, 25 °C, as a function of extent of binding (ν).
Figure 12. Thermodynamic parameters for the binding of dicloxacillin to human serum albumin, pH 7.4, 25 °C, as a function of extent of binding (νj).
SDS with BSA is associated with a conformational change in protein structure that exposes the hydrophobic amino acid residues from the protein interior that bind the alkyl chains of the SDS molecules.30 At pH 7.4 HSA is negatively charged (the isoelectric point of HSA is in the range 4.74.931) as are the drug molecules, so that their interaction with HSA would be expected to be largely hydrophobic and nonspecific and hence consistent with their tendency to form micelles. This suggests that the complex formation between HSA and the drugs, like SDS, will most probably involve a conformational change in protein structure. As for SDS-protein interaction,30 it is likely that some negatively charged drug molecules may bind specifically to cationic amino acid side chains (BSA has 98 such sites at pH 7 for SDS binding29), but the remaining drug binding will be nonspecific. The structure of the complexes cannot be deduced from thermodynamic data alone, but in view of the extent of drug binding with the protein, a “pearl necklace” model in which the polypeptide chain forms a string supporting micellar clusters of drug molecules is a possible model.32 (30) Tipping, E.; Jones, M. N.; Skinner, H. A. J. Chem. Soc., Faraday Trans. 1 1974, 70, 1306. (31) Bundschuh, I; Jacklemeyer, I.; Luneberg, E.; Bentzel, C.; Petzoldt, R.; Stolte, H. Eur J. Clin. Chem. Biochem. 1992, 30, 651. (32) Ibel, K.; May, R. P.; Kirschner, H.; Szadkowski, E.; Mascher, E.; Lundahl, P. Eur. J. Biochem. 1990, 190, 311.
νj
∆Gm° (kJ mol-1)
∆Hm° (kJ mol-1)
T∆Sm° (K kJ mol-1)
5 25 50 75 100
-14.7 -14.5 -14.3 -14.1 -13.9
cloxacillin -0.332 -0.287 -0.240 -0.201 -0.170
14.3 14.2 14.1 13.9 13.8
5 25 50 75 100
-21.1 -20.6 -20.0 -19.3 -18.7
dicloxacillin -0.219 -0.215 -0.210 -0.205 -0.201
20.9 20.4 19.8 19.1 18.5
5 25 50 75 100
-17.2 -17.0 -16.7 -16.5 -16.2
nafcillin -0.049 -0.048 -0.048 -0.047 -0.046
17.1 16.9 16.7 16.4 16.2
5 25 50 75 100
-15.7 -15.5 -15.3 -15.0 -14.8
flucloxacillin
Comparison of the values of ∆Gν, ∆Hν, and T∆Sν for the first 100 sites for all four drugs (Table 1) shows that binding strength as represented by the Gibbs energies of binding increases (becomes more negative) in the sequence dicloxacillin > nafcillin > flucloxacillin > cloxacillin, although the differences between the latter three are not large. The larger Gibbs energy of binding for the dichloro drug may relate to a contribution from dipole interactions that may be greater than in the case of the monohalide drugs, cloxacillin and flucloxacillin. The nafcillin interaction may be stronger than the monohalide interactions because of a greater hydrophobic contribution arising from the naphthalene ring. Summary The study shows that the structurally related penicillins bind extensively to human serum albumin at physiological pH and ionic strength. The binding isotherms of the drugs nafcillin and cloxacillin show maxima similar to those that have been observed in the binding of the anionic surfactant SDS to lysozyme.23 For the dicloxacillin-HSA system, the binding isotherm is biphasic and reflects the observation of two ccs for this drug. The binding isotherms have been used to obtain the Gibbs energies of drug binding per mole of drug, which in combination with the measured enthalpies of interaction for nafcillin, cloxacillin, and dicloxacillin from microcalorimetry, enabled the entropic contributions to the interaction to be determined. The thermodynamic parameters are consistent with strong hydrophobic interactions between the drugs and the protein, possibly resulting in the clustering of drug molecules (small micelles) around hydrophobic amino acid residues of the unfolded polypeptide chain. The complexes may be an example of the “pearl necklace” model as has been proposed in the case of protein-surfactant complexes. Acknowledgment. We thank the Xunta de Galicia for financial support and Fundacion Caixa Galicia for a grant for P.T. LA990538M
