A general, wide-rage 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 pharmacokinetic 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 in fluorescence upon binding at 37 degrees C, pH 7.4, the following dissociation constants were determined: Warfarin, 3.43 +/- 0.69 microM; dansylsulfonamide, 7.57 +/- 0.88 microM; and dansylsarcosine, 6.06 +/- 1.09 microM. Nonfluorescent ligands displace these probes competitively and the type of probe displaced identifies the site specificity of the ligands. Nonlinear least-squares analysis of the decrease in fluorescence accompanying the displacement yields the stoichiometry and the dissociation constant may also be estimated rapidly from displacement at a single competitor concentration. The method yields reliable Kd values for at least the range of 0.2 to 100 microM. Representative dissociation constants for the IIA site-specific ligands are as follows: phenylbutazone, 1.9 +/- 0.3 microM; U-99,499, 1.8 +/- 0.2 microM; U-96,988, 5.3 +/- 1.5 microM; and U-105,665, 42 +/- 7 microM. For the IIIA site we find the following Kd values: oxazepam, 27.7 +/- 2.1 microM; diazepam, 7.7 +/- 1.0 microM; and ibuprofen, 2.7 +/- 1.2 microM. The method is eminently suitable for large-scale screening.