MODIFICATION OF THE PROPERTIES OF BOVINE PANCREATIC RIBONUCLEASE A BY COVALENT ATTACHMENT OF D-GLUCONYL-GLYCINE RESIDUES*

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 K_m 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°. 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.