DETERMINATION OF THE MAGNITUDE AND ENANTIOSELECTIVITY OF LIGAND-BINDING TO RAT AND RABBIT SERUM ALBUMINS USING IMMOBILIZED-PROTEIN HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY STATIONARY PHASES

Rat, rabbit and human serum albumins were immobilized on an HPLC stati onary phase, and the resulting phases were tested for their abilities to determine the extent and enantioselectivity of ligand binding to th e respective albumins. A series of achiral and chiral compounds were c hromatographed on the phases including benzodiazepinones, non-steroida l anti-inflammatory drugs, amino acids, warfarin and leucovorin. The c hromatographic retentions of the benzodiazepinones and one series of n on-steroidal anti-inflammatory agents were compared with protein bindi ng data from ultrafiltration studies. The observed correlation factors (r) were consistently 0.999, indicating that the albumin phases can b e used to determine the magnitude of binding to the respective protein s. The enantioselectivity was also investigated, and the results indic ate that the stationary phases can be used to determine relative enant ioselectivities and intraspecies differences in this stereoselectivity . For example, when R- and S-warfarin were studied, R-warfarin was ret ained to a greater extent than S-warfarin by the rabbit serum albumin- stationary phase, whereas the opposite enantioselectivity was found fo r the rat and human albumins. Binding interaction studies were also co nducted on the rabbit and rat albumin stationary phases by sequentiall y adding increasing concentrations of octanoic acid to the chromatogra phic mobile phase. The octanoic acid reduced the retention of a series of non-steroidal anti-inflammatory agents, and the results of the exp eriments suggest that the interaction takes place at two or more sites on the albumin molecule and by anti-cooperative allosteric interactio ns and competitive displacement. The results of this study demonstrate that the immobilized serum albumin columns can be used to quantitate and probe ligand binding interactions.