CHARACTERIZATION OF ALKYLAMINE-SENSITIVE SITE IN ALPHA-2-MACROGLOBULIN

Methylamine reacts with the plasma protease inhibitor, α2-macroglobulin, to form an irreversible, covalent modification. Quantitation of the reaction indicates 3.9±(SD) 0.4 reactive sites per native tetrameric protein (M(r)=725,000) or one site per subunit. The reaction is selective and specific in that only 1 or 2 labeled peptides are observed on radioautography of peptide maps derived from [14C]methylamine-treated α2-macroglobulin. A single chymotryptic peptide was isolated in 56% overall yield from the labeled protein. The peptide sequence by Edman degradation was found to be Gly-Cys-Gly-Glu-X-Asn-Met(Val, Leu), Leu), in which X was the only radiolabeled phenylthiohydantoin derivative. Amino acid analysis and mass spectral analysis of the derivative suggests that X is γ-glutamylmethylamide. Because glutamic acid and glutamine residues do not normally react with alkylamines, this work presents presumptive evidence for an alternative activated center in selected proteins.