Reactions involving the amide and carboxyl groups of tobacco mosaic virus (TMV) protein

Abstract

Treatment of TMV with 3M hydroxylamine at pH 7.5 leads to a substituted product containing 21 bound hydroxylamine groups. Protein samples obtained by disruption of the virus in various ways participate in the reaction with hydroxylamine to a much smaller extent. The claim that the treatment of TMV with hydroxylamine at 60° leads to specific cleavage of a single peptide bond and exposes a reactive proline imino group could not be substantiated. Two approaches to the estimation of asparagine and glutamine residues in TMV-protein have been studied. The esterification of the free carboxyl groups and reduction of the protein ester was found to leave the amino acid amides intact. Subsequent hydrolysis of the protein derivative and estimation of the two dicarboxylic acids present provided data on the number of asparagine and glutamine residues originally present. The data indicate that TMV-protein of molecular weight 18,270 contains 21 amide residues per mole and that of these 12 ± 1 belong to asparagine and 9 ± 1 to glutamine. The second approach studied was the hydrazinolysis technique involving the differentiation of the resulting mono- and di-hydrazides of the dicarboxylic amino acids. This method, however, seems to be not useful for quantitative purposes because of the need for large correction factors. The data for the asparagine and glutamine content exceed by a large margin the amount of amide N present in the protein and are not in accord with the values from the reduction experiments.