REACTION OF 3-DIAZONIUM-1,2,4-TRIAZOLE WITH TRYPTOPHAN AT ACID PH - 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) reacted 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 increased exponentially from < 200 M-1 min-1 at pH 6 to > 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 caused loss in enzymatic activity at acidic and alkaline pH. Amino acid analysis 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 was developed. Using 3-diazonium-1,2,4-[5-14C]triazole, accurate tryptophan contents for trypsin, chymotrypsin, lysozyme, pepsin, insulin and fibrinogen were measured.