Antigenicity of Bovine Ribonuclease Modified at Tyrosine or Arginine Residues

Abstract

Bovine pancreatic ribonuclease was treated with tetranitromethane at a molar excess of 100 mol/mol protein at pH 8.0.Spectral studies showed that the nitration reaction can be better quantitated from the absorbance at 381 nm than from the generally used absorbance at 428 nm.With analytical isoelectric focusing it was found that the reaction product contained various derivatives, nitrated to a different extent.The two derivatives with the highest pI value were isolated by preparative isoelectric focusing in layers of granulated gel. Following performic acid oxidation, modified ribonuclease was digested successively with chymotrypsin and thermolysin. Amino acid analyses of the nitrated peptides showed that tyrosine‐115 was nitrated in the component with the highest pI value, while tyrosines‐76 and 115 were modified in the other nitrated component. The latter derivative exhibited the same enzymatic activity and antigenicity as unmodified ribonuclease.Bovine pancreatic ribonuclease was also modified with 1,2‐cyclohexanedione. After ion‐exchange chromatography and tryptic digestion of the oxidized protein it was determined that arginine residues 39 and 85 were modified for more than 90%, while arginine residue 10 was modified for 30%. Arginine residue 33 remained unchanged. The enzymatic activity of the cyclohexanedione‐treated ribonuclease was less than that of the unmodified protein and there was no loss of antigenic reactivity.It was concluded that tyrosine residues 76 and 115, and arginine residues 39 and 85 do not belong to antigenically relevant regions of bovine pancreatic ribonuclease.