Covalent binding between bucillamine derivatives and human serum albumin

Purpose. To clarify the mechanism of covalent binding between human serum albumin (HSA) and drags containing thiol groups, we studied the interactions between HSA and bucillamine (BA) and its derivatives. Methods. To determine the concentration of HSA-drug conjugate, we used columns of N-methylpyridium polymer cross-linked with ethylene glycol dimethacrylate (4VP-Me), and analyzed the reaction between HSA and BA derivatives kinetically. Following pseudo first-order reaction kinetics, the rate constants of reduction of non-mercaptoalbumin (HNA) to mercaptoalbumin (HMA) (k(a)) and formation of HSA-drug conjugate (k(c)) were determined. Results. Formation of HSA-drug conjugate was observed only for drugs containing one thiol group. In compound IV, the plots of k(a) and k(c) against pH were found to be linear. The HSA-drug conjugate was affected by various factors such as pK(a), pH, temparture and the microenviroment of Cys(34). The increases in k(a) and k(c) against pH were mainly due to the increase in mercaptide ion concentration. Further, fatty acid affected the microenviroment of Cys(34), which increased HSA-drug formation. Conclusions. Cys(34) located in a crevice on the surface of the protein plays an important role on the formation of HSA-drug conjugate. These results may be useful for elucidating the reaction mechanisms between various proteins and thiol compounds.