O-carboxamidomethyl tyrosine as a reaction product of the alkylation of proteins with lodoacetamide

Abstract

Iodoacetamide-1- 14C was found to be rapidly incorporated into RBP, a protein devoid of free SH, under conditions similar to those normally employed for the derivatization of SH. The reaction extent is pH dependent and at pH 10.0 one mole of acetamide was incorporated per mole of tyrosine residue. The amino acid composition of the alkylated RBP was found to be identical to RBP except for a loss of tyrosine and the appearance of a new dicarboxylic acid peak which was converted to tyrosine by prolonged acid hydrolysis Under similar reaction conditions, phenol and p-cresol reacted rapidly to form phenoxyacetamide and p-cresoxyacetamide. These phenolic ethers as well as anisole and phenetole were found to be readily hydrolized under the conditions normally used to hydrolyze protein. The incorporation of acetamide into RBP did not effect its riboflavinbinding capacity or its immunological reactivity to RBP antibody. The 14C alkylated RBP has been found to be a convenient tool for biological half-life studies. The tyrosine residues of glucagon react with iodoacetamide in a similar fashion and the use of 14C-iodoacetamide may prove to be a convenient means of introducing 14C into proteins. Iodoacetamide in the pH 7–10.0 range will derivatize the cysteine and tyrosine groups of proteins at comparable rates.