The reaction of 3-diazonium-1,2,4-triazole with tryptophan at acid pH: A scintillation method for tryptophan determination in proteins

Unlike conventional aryl diazonium reagents which are usually reactive at alkaline pH, 3-diazonium-1,2,4-triazole (3-DT) has been found to react with a variety of proteins at acid pH. Using indole, phenol, and imidazole as models of aromatic amino acid side chains, the rate of 3-DT coupling to the indole nucleus has been shown to increase exponentially from 2400 m −1 min −1 at pH 2.5. Coupling rates with phenol and imidazole show the conventional alkaline reactivity, with no evidence of reaction at acid pH. In parallel experiments using trypsin, 3-DT was found to cause loss in enzymatic activity at acidic as well as alkaline pH. Furthermore, amino acid analysis has demonstrated that the acidic reaction with trypsin resulted in the modification only of tryptophan. Based on the acidic specificity of 3-DT seen in these studies, a scintillation method for determining the tryptophan content of proteins has been developed. Using 3-diazonium-1,2,4-[5- 14C]triazole, accurate tryptophan contents for trypsin, chymotrypsin, lysozyme, pepsin, insulin, and fibrinogen have been measured.