A GENERAL, WIDE-RANGE SPECTROFLUOROMETRIC METHOD FOR MEASURING THE SITE-SPECIFIC AFFINITIES OF DRUGS TOWARD HUMAN SERUM-ALBUMIN

Binding of drugs to serum albumin is one of the most important pharmac okinetic determinants and the design of drugs should take advantage of this property. In the present work, the fluorescent ligands Warfarin and dansylsulfonamide were used as probes of IIA site of human albumin and dansylsarcosine as the probe of the IIIA site. From the changes i n fluorescence upon binding at 37 degrees C, pH 7.4, the following dis sociation constants were determined: Warfarin, 3.43 +/- 0.69 mu M; dan sylsulfonamide, 7.57 +/- 0.88 mu M; and dansylsarcosine, 6.06 +/- 1.09 mu M. Nonfluorescent ligands displace these probes competitively and the type of probe displaced identifies the site specificity of the Lig ands. Nonlinear least-squares analysis of the decrease in fluorescence accompanying the displacement yields the stoichiometry and the dissoc iation constants. The dissociation constant may also be estimated rapi dly from displacement at a single competitor concentration. The method yields reliable K-d values for at least the range of 0.2 to 100 mu M. Representative dissociation constants for the IIA site-specific Ligan ds are as follows: phenylbutazone, 1.9 +/- 0.3 mu M; U-99,499, 1.8 +/- 0.2 mu M; U-96,988, 5.3 +/- 1.5 mu M; and U-105,665, 42 +/- 7 mu M. F or the IIIA site we find the following K-d values: oxazepam, 27.7 +/- 2.1 mu M; diazepam, 7.7 +/- 1.0 mu M; and ibuprofen, 2.7 +/- 1.2 mu M. The method is eminently suitable for large-scale screening. (C) 1995 Academic Press, Inc.