ANALYSIS OF LOCAL ENVIRONMENT OF HISTIDINE RESIDUES IN PROTEINS BY HYDROGEN-TRITIUM EXCHANGE REACTION

Abstract

A general procedure to analyze local environment of individual histidine residues in proteins is presented. This procedure is based on the hydrogen-tritium exchange reaction at the C1 position of the imidazole ring of histidine. Since the exchange reaction is pH-dependent, dissociation constant (pKa) of the imidazole of a histidine residue can be measured from the plot of the rate constant versus pH of the reaction medium. Deviation of pKa of the histidine residue in question from its intrinsic pKa suggests that the neighboring charged group is interacting with the histidine. The magnitude of the exchange rate constant corresponds to solvent accessibility of the imidazole ring of the histidine in the three-dimensional structure of the protein. Using these correlations local environment of individual histidine residues in ribonuclease T1 and a serine protease produced by Streptomyces erythreus, whose three-dimensional structures are unknown, is discussed.