Effect of chemical glycosylation of RNase A on the protein stability and surface histidines accessibility in immobilized metal ion affinity electrophoresis (IMAGE) system.
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.
Baek
W O
WO
Laboratoire d'Interactions Moléculaires et de Technologie de Séparations, UPRES A 6022 CNRS, Université de Technologie de Compiègne, France.
Vijayalakshmi
M A
MA
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
D002234:Carbodiimides; D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D006639:Histidine; D012259:Ribonuclease, Pancreatic; D005944:Glucosamine