An automated flow injection-serial dynamic dialysis technique for drug-protein binding studies

Abstract

The interface of an automated flow injection analyzer with a dialysis unit to study drug-protein interactions using flow injection serial dynamic dialysis (FISDD) procedure is described. The method is based on the study of the kinetics of dialysis of the ligand in the absence and presence of protein. A study of the binding of sulfamethoxazole. sulfamethizole and sulfisoxazole to bovine serum albumin by means of such an automated system was undertaken to investigate the utility of FISDD technique for protein binding studies. The determination of dialysable sulfonamides was performed automatically by the FIA analyser. The influence of ionic strength and viscosity on the rate of dialysis was investigated. It was found that both variables did not affect the kinetic profile. Binding by the cellophane membrane was not encountered as a problem with the compounds studied. Binding parameters estimated for sulfamethoxazole were found to agree well with those reported in the literature. The Scatchard plots for the binding of sulfamethizole and sulfisoxazole with bovine serum albumin, revealed two classes of binding sites for each sulfonamide. The system was also used for the calculation of the dialytic rate constants. Experimental variables can be readily controlled to yield favoured conditions to study the protein binding phenomenon.