Spectrophotofluorometric evidence of negatively cooperative binding of warfarin and phenylbutazone to human serum albumin

Abstract

Competition between drugs for common binding sites on plasma proteins is an often discussed mechanism of drug interaction. In this study, the validity of the concept that phenylbutazone displaces warfarin from human serum albumin (HSA) by direct competition for the same sites was investigated. The fluorescence enhancement titration procedure of Kolb & Weber, Biochemistry 14 , 4471–4476 (1975) was used to characterise the warfarin-HSA interaction under the following conditions: excitation and emission wavelenghts - 310 & 390 nm respectively; buffer 0.1 M phosphate, pH 7.0 (25°C) and [HSA] - 10 −6M. The titrations were then repeated in the presence of varying concentrations of phenylbutazone (10 −6M − 5 × 10 −4M). Bjerrum plots of the data showed a shift of the warfarin-HSA binding curves to higher free levels of warfarin with increasing concentrations of phenylbutazone. However this shift was saturable, contrary to expectation for directly competitive antagonism. Together with indirect evidence from the literature, the results suggest that displacement of warfarin by phenylbutazone is via a negatively cooperative mechanism rather than by direct competition.