Optimal conditions for the use of protein l-isoaspartyl methyltransferase in assessing the isoaspartate content of peptides and proteins

Protein l-isoaspartyl methyltransferase provides a basis for enzymatic measurement of atypical, isoaspartyl linkages which make a major contribution to protein microheterogeneity. The low V max of the methyltransferase reaction and the instability of the methyl ester can hinder accurate determinations, and different laboratories using different conditions have achieved discrepant values for the isoaspartate content of the same proteins. To investigate the effects of these conditions, and to optimize the assay, isoaspartyl δ sleep-inducing peptide was methylated under a variety of conditions. We found that 1 μ m methyltransferase was required to obtain stoichiometric modification of 2 μ m peptide in 40-min reactions at pH 6.2 and 30°C. A computer model utilizing kinetic constants obtained from studies on initial rates of methylation predicted the same requirement for enzyme concentration. Carrier protein was necessary for optimal methyltransferase activity at enzyme concentrations below 0.4 μ m. Stoichiometric methylation required concentrations of S-adenosylmethionine to be in substantial excess over those of peptide; 50 μ m S-adenosylmethionine is the minimum needed for complete modification of 10 μ m peptide. Spontaneous demethylation was significant under all conditions tested, so that the methyl ester itself never reached a ratio of 1 mol/mol of total peptide. These results demonstrate that the most accurate measurements of isoaspartate are obtained when reactions are carried out at low peptide concentrations, high S-adenosylmethionine concentrations, and high enzyme concentrations. Moreover, quantitation will be significantly better when based on measurements of both methanol and methyl esters rather than on measurements of intact methyl esters alone.