Multiple Carboxymethylation of Histidines in Bovine Ribonuclease A

Reaction of RNAase A with bromoacetate at pH 5.5 for 1–42 days results in multiple reactions. Alkylation of residues proceeds in the sequence: (1) N‐1 of histidine‐119; (2) methionine (probably methionine‐30); (3) N‐3 of histidine‐12; (4) N‐3 of histidine‐105 and N‐3 of 1‐carboxy‐methyl histidine‐119; and (5) lysine‐1. Both histidine‐12 and histidine‐119 of the same active site are carboxymethylated. A derivative carboxymethylated at both active site histidines is obtained in 1 day and probably some of this derivative is obtained in short reaction times. This is contrary to the conclusions of earlier investigations. Histidine‐48 undergoes little or no reaction. The results are in accord with the X‐ray structure of RNAase.