Effects of Drug Binding on the Esterase-Like Activity of Human Serum Albumin. IV. Application of an Analog Computer to Determination of the Multiple Dissociation Constants

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The inhibition parameters of various drugs (inhibitor, I) for the reaction of p-nitrophenyl acetate (substrate, S) with human serum albumin (HSA) could not be directly determined, because HSA has two esterase-like active sites for S (i.e. R and T sites) and three drug binding sites (i.e. R, T and U sites) [Ozeki et al., Chem. Pharm. Bull., 28, 525 (1980)]. In the present study, the dissociation constants between I and the individual binding sites on HSA, K_{I, R}, K_{I, T} and K_{I, U}, were determined by analog computer simulation of the inhibition. Clofibric acid, ibuprofen and dansylsarcosine were found to bind solely to the R site, and their K_{I, R} values were determined. Flufenamic acid, ethacrynic acid and salicylic acid bind primarily to the R site and then to the T site, and their K_{I, R} and K_{I, T} values were estimated. The K_{I, U} and K_{I, R} values for phenylbutazone and sulfinpyrazone were also determined, and their K_{I, U} values were smaller than their K_{I, R} values. It is likely that warfarin binds primarily to two binding sites unconcerned with the esterase-like activities and then to the R site of HSA, and these three dissociation constants were estimated. Since this kinetic method distinguishes the drug binding sites on HSA and gives the dissociation constants for the individual sites, it is very useful for studies on drug interaction with HSA.