ENANTIOSELECTIVITY OF BOVINE SERUM ALBUMIN-BONDED COLUMNS PRODUCED WITH ISOLATED PROTEIN-FRAGMENTS .2. CHARACTERIZATION OF PROTEIN-FRAGMENTS AND CHIRAL BINDING-SITES

Enantioselectivity of bovine serum albumin (BSA)-bonded columns produc ed with isolated protein fragments has been investigated. The BSA frag ment, BSA-FG75, was isolated by size exclusion chromatography followin g peptic digest of BSA. The isolated BSA-FG75 was further fractionated to two fractions, BSA-F1 and BSA-F2, by anion-exchange chromatography . BSA-F1 and BSA-F2 had molecular mass of about 35 000 daltons, estima ted by matrix-assisted laser desorption ionization time-of-flight (MAL DI-TOF) mass spectrometry. BSA-F1 has amino acid residues 1-307 estima ted by electrospray ionization (ESI) mass spectrometry, while BSA-F2 i s an N-terminal half BSA fragment. The BSA, BSA-FG75, BSA-F1 and BSA-F 2 proteins were bound to aminopropyl-silica gels activated by N,N'-dis uccinimidyI carbonate. The bound amounts of the BSA fragments were 2.2 -2.7 times more than that of the intact BSA. Chiral recognition of 2-a rylpropionic acid derivatives, benzodiazepines, warfarin and benzoin w as obtained with the BSA fragment-bonded columns. The non-enantioselec tive interactions of benzoin and benzodiazepines except for clorazepat e with BSA fragments were increased with protein surface coverages, wh ile those of 2-arylpropionic acid, clorazepate and warfarin were decre ased. The BSA fragment columns gave higher enantioselectivity for lora zepam and benzoin, and lower enantioselectivity for other compounds te sted, compared with the BSA column. These results might be due to chan ges in the globular structure of the BSA fragment and/or changes in th e local environment around the binding sites.