title: Development of tryptophan-modified human serum albumin columns for site-specific studies of drug-protein interactions by high-performance affinity chromatography.
abstract: 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 examined the changes in chromatographic properties that occur for immobilized HSA following the chemical modification of HSA's lone tryptophan residue (Trp-214). Trp-214 was reacted with o-nitrophenylsulfenyl chloride, followed by immobilization of the modified protein and normal HSA onto separate silica-based HPLC supports. The binding properties of the modified and normal HSA were then analyzed and compared by using frontal analysis and zonal elution experiments employing R/S-warfarin and L-tryptophan as probe compounds for the warfarin and indole binding regions of HSA. The modified HSA was found to have the same number of binding sites as normal HSA for R-warfarin and L-tryptophan but lower association equilibrium constants for these test solutes. Zonal elution studies with R- and S-warfarin on the modified HSA column demonstrated 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.
authors.affiliation: Chemistry Department, University of Nebraska, Lincoln 68588-0304, USA.
authors.forename: A
authors.initials: A
authors.lastname: Chattopadhyay
authors.forename: T
authors.initials: T
authors.lastname: Tian
authors.forename: L
authors.initials: L
authors.lastname: Kortum
authors.forename: D S
authors.initials: DS
authors.lastname: Hage
mesh_terms: D001032:Apazone / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D011485:Protein Binding; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry*; D014859:Warfarin / Q000378:metabolism*
chemical_list: D012709:Serum Albumin; D014859:Warfarin; D014364:Tryptophan; D001032:Apazone