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

Abstract

The proton magnetic resonance spectrum at 300 MHz of the histidine residues in a semisynthetic derivative of bovine pancreatic ribonuclease (RNase A) has been determined. The derivative RNase 1-118 . 111-124 was prepared by enzymically removing six 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 ribonuclease (RNase 111-124) [Lin, M.C., Gutte, B., Moore, S., & Merrifield, R.B. (1970) J. Biol. Chem. 245, 5169-5170]. 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-119 was confirmed by the use of a selectively deuterated derivative. The titration behavior of all four histidine residues is indistinguishable from that observed by others for bovine pancreatic ribonuclease A. Partial dissociation of the noncovalent semisynthetic complex was evident at 30 degrees 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 X 10(3) M-1.

Histidine pKa shifts accompanying the inactivating Asp121----Asn substitution in a semisynthetic bovine pancreatic ribonuclease.

“…The C2 proton NMR spectrum of semisynthetic RNase-(1-118). (111-124) and its pH dependence also have been obtained (15). The titration behavior of the four histidine residues in this semisynthetic derivative was indistinguishable from that found by others for RNase A.…”

“…Spectrum A of Fig. 1 (15). However, in this semisynthetic complex, there is a fifth resonance (stippled resonance in spectra B-D of Fig.…”

“…RNase-(1-118) was prepared by the successive digestion ofRNase A with pepsin and carboxypeptidase A (15), except that the gel-filtered preparations were further purified by isocratic ion-exchange chromatography at 50C on SP-Sephadex G-25 (40-to 120-,um particles; Pharmacia) in 0.13 M sodium phosphate, pH 6 (19,20) and purified by methods previously described (15). NMR Experiments.…”

Structural investigation of catalytically modified F120L and F120Y semisynthetic ribonucleases

“…Preparation of RNase RNase 1-118 was prepared according to previously published procedures (Doscher et al, 1983b), except that the gel-filtered product was further purified by isocratic ion-exchange chromatography at 5 "C on SP-Sephadex G-25,40-120 pm (0.13 M Na phosphate, pH 6.65). Stock solutions of RNase 1-1 18 were characterized by amino acid analysis of acid hydrolysates of aliquot samples and by the determination of specific activity against cytidine 2',3'-cyclic phosphate, both in the absence and in the presence of a saturating level of RNase 11 1-124.…”

“…The tetradecapeptide analogs were synthesized by the Wayne State University Macromolecular Core Facility and purified by methods previously described (Sasaki et al, 1979;Doscher et al, 1983b). Stock solutions of the peptides were characterized and standardized by amino acid analysis of acid hydrolysates of aliquot samples.…”