Characterization of the histidine proton nuclear magnetic resonances of a semisynthetic ribonuclease

Abstract

The PMR spectrum at 300 MHz of the histidine residues in a semisynthetic derivative of bovine pancreatic RNase A was determined. The derivative RNase 1-118 .cntdot. 111-124 was prepared by enzymically removing 6 residues from the COOH terminus of the protein (positions 119-124) and then complementing the inactive RNase 1-118 with a chemically synthesized peptide containing the COOH-terminal 14 residues of RNase (RNase 111-124). Comparison of the line positions of the C(2)-1H resonances of these residues and of their pH dependence with those reported by other workers has allowed assignment of the resonances to individual residues, as well as the determination of individual pK values for histidine-12, histidine-105 and histidine-119. The assignment of histidine-12, histidine-105 and histidine-119. The assignment of histidine-119 was confirmed by the use of a selectivity deuterated derivative. The titration behavior of all 4 histidine residues is indistinguishable from that observed by others for bovine pancreatic RNase. A partial dissociation of the noncovalent semisynthetic complex was evident at 30.degree. C, pH 4.0, 0.3 M NaCl; pertinent spectra were analyzed to provide an estimate of the association constant between the component chains under these conditions of 1.9 .times. 103 M-1.