Structure-function relationship in glycosylated α-chymotrypsin as probed by IMAC and IMACE

• Published in: Biochimica et biophysica acta Vol. 1433; no. 1; pp. 198 - 209
• Main Authors: Jiang, Kai-You, Pitiot, Olivier, Anissimova, Marya, Adenier, Herve, Vijayalakshmi, Mookambeswaran A.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 17.08.1999
• Subjects: Animals; Bovine chymotrypsin; Capillary electrophoresis; Caseins - chemistry; Cattle; Chemical glycosylation; Chromatography, Affinity - methods; Chymotrypsin - chemistry; Electrophoresis, Capillary - methods; Glucosamine - chemistry; Glycosylation; Histidine - chemistry; Histidine accessibility; Hydrogen Bonding; Imino Acids - chemistry; Immobilized metal-ion affinity chromatography; Mass Spectrometry - methods; Metals; Structure-Activity Relationship; Tyrosine - analogs & derivatives; Tyrosine - chemistry; Chemical glycosylation; Capillary electrophoresis; IMA, immobilized metal-ion affinity; BTEE, N-benzoyl- l-tyrosine ethyl ester; IEF, isoelectric focusing; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; IMAC, immobilized metal-ion affinity chromatography; IMACE, immobilized metal-ion affinity capillary electrophoresis; Histidine accessibility; ESI-MS, electrospray ionization mass spectrometry; Immobilized metal-ion affinity chromatography; IDA, iminodiacetic acid; Bovine chymotrypsin
• ISSN: 0167-4838; 0006-3002; 1879-2588; 1878-2434
• DOI: 10.1016/S1388-1981(99)00087-6

Chemical glycosylation of bovine α-chymotrypsin, by a glucosamine adduct on the carboxyl group, results in the modification of its catalytic activity. The structural alterations of α-chymotrypsin resulting from its glycosylation are studied by immobilized metal-ion affinity chromatography (IMAC) and immobilized metal-ion affinity capillary electrophoresis (IMACE). The chemical glycosylation of α-chymotrypsin generates two distinct subpopulations of the protein: one which totally loses the initial affinity for IDA-Cu(II) and another which exhibits an increased affinity for the metal chelate ligand.