title: Effect of chemical glycosylation of RNase A on the protein stability and surface histidines accessibility in immobilized metal ion affinity electrophoresis (IMAGE) system.
abstract: Immobilized metal ion affinity gel electrophoresis (IMAGE) has been applied to study the change of the surface histidines topography of RNase A when chemically glycosylated on exposed carboxylic groups with glucosamine using carbodiimide as cross-linker, under mild conditions. Two populations of glycosylated RNase A, one with a single glucosamine and another with two glucosamine attached, were obtained. These chemically glycosylated RNase A conserved about 80% of native enzymatic activity and their pls were increased in comparison to native RNase A. The chemically glycosylated RNase A showed dramatic enhancement for thermal stability, while proteolytic resistance was similar to that of native RNase A. Chemically glycosylated RNase A showed a slightly increased affinity to IDA-Cu(II) as compared to the native enzyme, which indicates that a conformational change and/or a decrease in steric hindrance around accessible surface histidines (His 12 or His 119 and His 105) has occured. IMAGE is a useful method to analyse subtle conformational changes in proteins which result in subtle changes in histidine accessibilities.
authors.affiliation: Laboratoire d'Interactions Moléculaires et de Technologie de Séparations, UPRES A 6022 CNRS, Université de Technologie de Compiègne, France.
authors.forename: W O
authors.initials: WO
authors.lastname: Baek
authors.forename: M A
authors.initials: MA
authors.lastname: Vijayalakshmi
mesh_terms: D000818:Animals; D002234:Carbodiimides; D002417:Cattle; D003432:Cross-Linking Reagents; D004586:Electrophoresis / Q000379:methods; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism*; D005944:Glucosamine / Q000378:metabolism*; D006031:Glycosylation; D006639:Histidine; D006358:Hot Temperature; D007700:Kinetics; D008970:Molecular Weight; D011489:Protein Denaturation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism*; D018629:Spectrometry, Mass, Secondary Ion; D013499:Surface Properties; D013816:Thermodynamics
chemical_list: D002234:Carbodiimides; D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D006639:Histidine; D012259:Ribonuclease, Pancreatic; D005944:Glucosamine