States of amino acid residues in proteins: IX. Histidine residues in ribonuclease in the presence of cytidine- and uridine-3′-phosphates as observed with diazonium-1-H-tetrazole

Abstract

Diazonium-1-H-tetrazole (DHT), a new coupling reagent for histidine residues, was reacted with bovine pancreatic ribonuclease (RNase) in the presence and absence of nucleotides; cytidine-3′-phosphate (CMP), uridine-3′-phosphate(UMP), adenosine-3′-phosphate (AMP), and guanosine-3′-phosphate (GMP). Coupling to the four histidine residues in the native RNase molecule in the absence of nucleotide proceeds in four steps with increasing DHT concentration, such that each of these four residues is coupled at a different DHT concentration. The reactivities were affected by the presence of CMP or UMP, a product of the RNase-catalyzed reaction, whereas AMP or GMP, which are not products of the reaction, show no effect on the reactivities. The effect of CMP is different from that of UMP; CMP enhances the reactivity of a residue which is the third to be coupled in the absence of nucleotide, while UMP enhances the reactivity of a residue which is the second or fourth to be coupled. The possible participation in the enzymic activity of these two histidine residues with different reactivities and their different responses to nucleotides were thus demonstrated.