STATES OF AMINO ACID RESIDUES IN PROTEINS. 3. HISTIDINE RESIDUES IN INSULIN, LYSOZYME, ALBUMIN AND PROTEINASES AS DETERMINED WITH A NEW REAGENT OF DIAZO-I-H-TETRAZOLE.

Abstract

A new coupling reagent, diazonium- i -H-tetrazole, for spectrophorometric determination of histidine residues in proteins was explored and applied to several proteins. The reagent has the following characteristics advantageous to the determination and is superior to common diazonium compounds. (a) Histidinebisazo- i -H-tetrazole, which is the reaction product to be determined spectrophotometrically, has a strong absorption band at 480 mμ, while tyrosinebisazo- i -H-tetrazole has a weaker band at a considerably longer wavelength, 550 mμ. (b) The coloration due to the formation of biazohistidine residues proceeds to completion before the bis coupling to tyrosine residues. (c) The reagent does not afford colored by-products during its reactions with amino acids or proteins at alkaline pH, so that one can use a high concentration of reagent necessary to determine the end point of coloration. Because of these characteristics, one can determined the molar histidine content simply by absorption photometry at 480 mμ of reaction mixtures of protein and the reagent. The reagent reacts uniformly with all of the histidine residues in bacitracin, lysozyme (EC 3.2.1.17), insulin, albumin and trypsin (EC 3.4.4.4). On the other hand, the reactions with the histidine residues in trypsinogen, chymotrypsinogen and α-chymotrypsin (EC 3.4.4.5) proceed in two steps, in which the reaction in the second step at higher reagent concentrations is due to the coupling to bound histidine residues. The moles of bound residues in the latter proteins were determined and discussed referring to the data obtained previously with other reagents.