Influence of albumin concentration, albumin species, pH and temperature on the stereoselectively different binding of (S)-(+)- and (R)-(-)-1-methyl-5-phenyl-5-propyl-barbiturate to serum albumin.

Using normal conditions (phosphate buffer pH 7.4, room temperature) and 1% human serum albumin (HSA), (R)-(-)-1-methyl-5-phenyl-5-propyl-barbiturate = (R)-(-)-1) as compared to (S)-(+)-1 was preferentially bound to albumin: the difference in the enantioselective binding was large (factor of 2) with low substrate concentrations and much smaller (factor of 1.3) with high substrate concentrations (range 5-620 micrograms/ml); with (S)-(+)-1 the decrease of the percent binding values observed with increasing concentrations was only moderately marked. The stereoselective binding difference ((R)-(-)- being greater than (S)-(+)-1) was always present independent of the albumin concentration (range 0.5-10%), the pH value (range 5-10) and the temperature (range 5-50 degrees C). Using 20 micrograms/ml as substrate concentration the stereospecific binding difference was large in solutions with low HSA concentration, in acidic and neutral pH range, and in experiments with low temperature. In contrast to HSA, bovine as well as rabbit serum albumin bound (S)-(+)-1 to a higher percentage than (R)-(-)-1; using rat serum albumin an enantioselective binding difference was not obtained. Taking into account for each of three mammalian albumins the respective number of binding classes and sites in each class, the affinity constant and total binding constant, it becomes evident that they bind (S)-(+)- and (R)-(-)-1 quantitatively differently in a species-specific manner.