Binding of bupivacaine to human serum proteins, isolated albumin and isolated alpha-1-acid glycoprotein. Differences between the two enantiomers are partly due to cooperativity.

Binding parameters of R(+)- and S(-)-bupivacaine were determined for human serum proteins, human alpha-1-acid glycoprotein (AAG) and human serum albumin (HSA), using ultrafiltration. Binding parameters were estimated according to the Scatchard model of the law of mass action using nonlinear regression. A sigmoid (cooperativity) term was added when needed. Both enantiomers exhibited a two site binding profile for human serum and for a solution containing AAG and HSA at physiological concentrations. At concentrations lower than 40 microM (concentrations encountered in clinical situations), the low capacity, high affinity apparent site was predominant and S(-)-bupivacaine exhibited a higher free fraction than R(+)-bupivacaine. At concentrations higher than 60 microM, the opposite situation was observed and the S(-) enantiomer showed much higher binding to AAG than the R(+) enantiomer. Two cooperativity phenomena occurred. Negative cooperativity was observed when AAG and HSA were combined in the same solution. S(-) and R(+) enantiomers exhibited different behavior toward purified AAG and HSA due in part to complex allosteric cooperativity (positive or negative depending on the ligand/protein ratio). In conclusion, we observed stereoselective binding of bupivacaine to AAG and HSA. Moreover, cooperativity occurred, and the behavior of the two enantiomers showed marked differences in this respect.