S-Methylmethionine-29 Ribonuclease A: I. Preparation and Proof of Structure

Abstract

One of the 4 methionine residues in ribonuclease A, the one at position 29, is exposed to the solvent when the protein is in its native conformation. Under such conditions, this residue can be alkylated by CH3I to yield a fully active monomethyl derivative. The rate of alkylation, measured chromatographically by the disappearance of ribonuclease A, is constant between pH 2.5 and pH 5.0. At pH 2.5, ribonuclease A is converted quantitatively into S-methylmethionine-29 ribonuclease, which can be separated by chromatography on IRC-50. The structure of the methylated protein was established by isolating and characterizing peptides derived from the 14C-labeled methyl derivative after enzymatic and chemical cleavage. Key techniques were chemical cleavage with cyanogen bromide, followed by isolation of the labeled peptides on Sephadex rather than by ion exchange chromatography. Since minor components are not overlooked in a separation based only on size, the confidence with which it can be said that S-methylmethionine-29 ribonuclease is a homogeneous derivative rests primarily on the specific radioactivity of the peptide containing methionine-29, irrespective of yield. At least 92% of the molecules of ribonuclease in the chromatographic peak are alkylated on methionine-29.