On the use of tetranitromethane as a nitration reagent for tyrosyl residues

Abstract

Nitration with tetranitromethane has proven to be an exceptionally valuable means for studying residues of proteins [ 1, 2] . The agent can be employed under mild conditions i.e. room temperature at low concentrations and at pH values close to or at 8.0. The reaction is generally terminated by gel-filtration at the same pH [ 1] . In several reports the sum of the nitrotyrosyl and tyrosyl content of the nitrated protein was in good agreement with the tyrosyl content of the starting material. However, it was noted recently in a few cases [3-51 that the amount of nitrated tyrosine plus tyrosine was less than expected. The main difference between the original reports and the reports concerning the destruction of tyrosyl residues is in the procedure employed to terminate the reaction. In the past excess reagent was removed at pH 8.0 as initially recommended. Loss of tyrosine was observed in instances where nitration was stopped by lowering the pH. As part of a study to delineate further the specificity of TNM toward proteins [6, 71, we have now investigated the possible consequences of this latter procedure. Ribonuclease A has been used as a model since it does not contain tryptophanyl or cysteinyl residues which might add additional complications [6,7].