DEVELOPMENT OF TRYPTOPHAN-MODIFIED HUMAN SERUM-ALBUMIN COLUMNS FOR SITE-SPECIFIC STUDIES OF DRUG-PROTEIN INTERACTIONS BY HIGH-PERFORMANCE AFFINITY-CHROMATOGRAPHY

Human serum albumin (HSA) is one of the main proteins involved in the binding of drugs and small solutes in blood or serum. This study exami ned the changes in chromatographic properties that occur for immobiliz ed HSA following the chemical modification of HSA's lone tryptophan re sidue (Trp-214). Trp-214 was reacted with o-nitrophenylsulfenyl chlori de, followed by immobilization of the modified protein and normal HSA onto separate silica-based HPLC supports. The binding properties of th e modified and normal HSA were then analyzed and compared by using fro ntal analysis and zonal elution experiments employing RIS-warfarin and L-tryptophan as probe compounds for the warfarin and indole binding r egions of HSA. The modified HSA was found to have the same number of b inding sites as normal HSA for R-warfarin and L-tryptophan but lower a ssociation equilibrium constants for these test solutes. Zonal elution studies with R- and S-warfarin on the modified HSA column demonstrate d the importance of Trp-214 in determining the stereoselective binding of HSA for these agents. These studies also indicated that tryptophan modification can alter HSA-based separations for chiral solutes. (C) 1998 Elsevier Science B.V. All rights reserved.