A competitive labelling method for determining the ionization constants and reactivity of individual histidine residues in proteins. The histidines of α-chymotrypsin

Abstract

A competitive labelling method (Kaplan et al., 1971), using tritiated 1-fluoro-2,4-dinitrobenzene as the labelling reagent, is described for determining the ionization constants and reactivities of individual histidine residues in proteins. When this method was applied to the two histidines of α-chymotrypsin, histidine-57 was found to have pKa 6.8 and a reactivity ten times that of α-N-acetyl-l-histidine. Histidine-40 had pKa 6.7 and a reactivity approximately six times that of α-N-acetyl-l-histidine. Between pH7.5 and 8 the reactivities of both histidines decrease simultaneously to approximately that of α-N-acetyl-l-histidine. The high reactivities of the histidines are attributed to hydrogen bonding, which increases the nucleophilicity of the imidazole ring. The sharp decrease in reactivity between pH7.5 and 8 is attributed to a conformational change that disrupts the hydrogen bonding by these residues. The reactivity data support the proposal of a charge-relay mechanism involving histidine-57 (Blow et al., 1969), which makes serine-195 more nucleophilic but indicates that this system is fully operative only in the enzyme–substate complex.