title: Modification of the properties of bovine pancreatic ribonuclease A by covalent attachment of D-gluconyl-glycine residues.
abstract: Bovine pancreatic ribonuclease A was reacted with D-gluconyl-glycine azide in aqueous solution at pH 8.9, in absence of phosphates. Five out of 11 amino groups can be reproducibly modified and the penta D-gluconyl-glycinated ribonuclease A had greater than 70% of the enzymic activity of the unmodified enzyme toward cytidine 2', 3'-cyclic phosphate as well as uridine 2', 3'-cyclic phosphate and yeast RNA. The kinetic parameters Km and k2 of the modified enzyme were calculated from double-reciprocal Lineweaver-Burk plots, using cytidine 2', 3'-cyclic phosphate as the substrate. The native and chemically modified protein exhibited identity in their reversible thermal transitions at neutral pH, with midpoint at about 60 degrees. Circular dichroism measurements indicated that the overall conformation of the D-gluconyl-glycinated enzyme is not significantly different from that of the unglycosylated parent enzyme. The modified protein was less sensitive than the native ribonuclease A to attack by chymotrypsin, pepsin and elastase, indicating a protecting effect of the D-gluconyl-glycine units. Similar properties are shown by the glycosylated bovine pancreatic ribonuclease B.
mesh_terms: D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D005942:Gluconates; D005998:Glycine / Q000031:analogs & derivatives*; D006023:Glycoproteins; D007202:Indicators and Reagents; D010179:Pancreas / Q000201:enzymology; D011487:Protein Conformation; D012260:Ribonucleases / Q000138:chemical synthesis*
chemical_list: D005942:Gluconates; D006023:Glycoproteins; D007202:Indicators and Reagents; C025228:D-gluconyl-glycine azide; D012260:Ribonucleases; D005998:Glycine