Enantioselectivity of bovine serum albumin-bonded columns produced with isolated protein fragments. II. Characterization of protein fragments and chiral binding sites.
Enantioselectivity of bovine serum albumin (BSA)-bonded columns produced with isolated protein fragments has been investigated. The BSA fragment, BSA-FG75, was isolated by size exclusion chromatography following 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 35000 daltons, estimated by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, BSA-F1 has amino acid residues 1-307 estimated by electrospray ionization (ESI) mass spectrometry, while BSA-F2 is an N-terminal half BSA fragment. The BSA, BSA-FG75, BSA-F1 and BSA-F2 proteins were bound to aminopropyl-silica gels activated by N,N'-disuccinimidyl 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-arylpropionic acid derivatives, benzodiazepines, warfarin and benzoin was obtained with the BSA fragment-bonded columns. The non-enantioselective interactions of benzoin and benzodiazepines except for clorazepate with BSA fragments were increased with protein surface coverages, while those of 2-arylpropionic acid, clorazepate and warfarin were decreased. The BSA fragment columns gave higher enantioselectivity for lorazepam and benzoin, and lower enantioselectivity for other compounds tested, compared with the BSA column. These results might be due to changes in the globular structure of the BSA fragment and/or changes in the local environment around the binding sites.
Haginaka
J
J
Faculty of Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya, Japan.
Kanasugi
N
N
D001569:Benzodiazepines / Q000302:isolation & purification; D001573:Benzoin / Q000302:isolation & purification; D001665:Binding Sites; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003009:Clorazepate Dipotassium / Q000302:isolation & purification; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011422:Propionates / Q000302:isolation & purification; D017434:Protein Structure, Tertiary; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013237:Stereoisomerism; D014859:Warfarin / Q000302:isolation & purification
D010446:Peptide Fragments; D011422:Propionates; D001569:Benzodiazepines; D012710:Serum Albumin, Bovine; D014859:Warfarin; D003009:Clorazepate Dipotassium; D010434:Pepsin A; D001573:Benzoin