The Nature of the Amino Acid Residues Involved in the Inactivation of Ribonuclease by Iodoacetate

SUMMARY Ribonuclease is inactivated by iodoacetate at 40”, the rate depending upon the pH at which reaction is carried out. It, is most rapid between pH 5.5 and 6, and at, pH 2.8, less rapid at intermediate pH values and under more alkaline conditions (pH 8.5 to 10). The variation in rate is a consequence of the fact that different types of amino acid residues in the ribonuclease molecule react with iodoacetate at the different pH values. Amino acid analyses showed that at pH 5.5 to 6, a histidine residue is involved, at pH 8.5 to pH 10 reaction occurs with the e-amino groups of lysine residues, whereas at pH 2.8, the thio- ether sulfur of one or more methionine residues is alkyla,ted. After removal of iodoacetate, the various reaction mixtures were chromatographed on columns of IRC-50 in an attempt to isolate purified carboxymethylated derivatives. From the mixture present after reaction at pH 5.5, it was possible to isolate a chromatographically homogeneous protein that was totally in- active and differed from ribonuclease A only in that the imidaz- ole group of a single histidine residue had been carboxymethyl- ated. The fact, that the pH optimum for the reaction leading to the formation of imidazole carboxymethyl ribonuclease is around pH 5.5 to 6, instead of at the more alkaline pH values observed for the alkylation of cy-N-acetylhistidine, suggests that inactivation takes place as a result of substitution on a particular one of the four histidine residues present in ribonuclease.