Spectroscopic techniques in the study of protein binding. A fluorescence technique for the evaluation of the albumin binding and displacement of warfarin and warfarin-alcohol.

Abstract

1. The binding of racemic mixtures of warfarin and warfarin-alcohol to human serum albumin (HSA) is accompanied by an increase in the fluorescence quantum yield of these compounds. This property has been used to measure the characteristics of the binding of warfarin and warfarin-alcohol to HSA at 22 degrees C and 37 degrees C. Within the limits of the technique, no significant differences between the number of binding sites and strength of binding at the tight site at either temperature were observed. 2. The fluorescence of warfarin and warfarin-alcohol was used to label their binding site on HSA and to study the effects of other drugs on their binding. The results indicate that these two molecules are bound to the same site on HSA. 3. The validity of using changes in the fluorescence of warfarin as a measure of its displacement from HSA was investigated. Good correlations were observed between drug-induced decreases in the fluorescence of bound warfarin and displacement as measured by equilibrium dialysis. The displacement of warfarfin, as detected by fluorescence, correlates well with the increase in free warfarin resulting from addition of therapeutic drug concentrations to undiluted human serum. 4. The most potent displacing agents, by all the methods used, were iophenoxic acid, phenylbutazone and oxyphenylbutazone. The first of these is no longer used clinically, but the latter two are and have been reported to cause hypoprothrominaemia by displacing warfarin from HSA. The present study indicates that changes in the fluorescence of warfarin bound to HSA can be used to measure displacement of bound warfarin and to screen drugs that may cause clinically significant interactions by this mechanism.