title: Covalent binding of benzoquinone to reduced ribonuclease. Adduct structures and stoichiometry.
abstract: As a model for the reaction of chemically reactive quinone metabolites with cellular proteins in vivo, the reactions of benzoquinone (BQ; 1-64 mol/mol of protein) with bovine pancreatic ribonuclease A (RNase), reduced RNase, S-(amidomethylated) reduced RNase, and reduced guanidinated RNase were investigated. The reaction stoichiometry and products were characterized by means of HPLC, UV-vis spectrophotometry, electrospray mass spectrometry, amino acid analysis, alkaline permethylation analysis, and measurement of the covalent binding of [14C]BQ to protein. Native RNase and S-(amidomethylated) reduced RNase show no reaction with BQ over 60 min at pH 7.4-9.6, whereas reduced RNase, which has 8 free SH groups/mol, reacts rapidly with exactly 24 molecules of BQ, of which 8 become covalently bound to protein-SH groups while 16 are reduced to hydroquinone. Half of the latter is formed via BQ oxidation of the initially formed S-(2,5-dihydroxyphenyl)cysteine moieties. Michael addition of a second protein nucleophile to each resulting S-(2,5-quinoyl)cysteine moiety, followed by reoxidation of that addition product by BQ, generates the second group of 8 molecules of HQ and results in cross-linking. Reduced guanidinated RNase, in which most of the lysines are blocked by guanidination with O-methylisourea, also reacts rapidly with BQ, but only ca. 16 equiv are consumed; of these, 8 become covalently bound to protein-SH groups while the others are reduced to HQ. Thus, even though the lysine residues in native RNase and S-(amidomethylated) reduced RNase do not react with BQ, they may react with (2,5-quinonyl)-S-protein moieties.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Medicinal Chemistry, University of Kansas, Lawrence 66045-2506.
authors.forename: R P
authors.initials: RP
authors.lastname: Hanzlik
authors.forename: S P
authors.initials: SP
authors.lastname: Harriman
authors.forename: M M
authors.initials: MM
authors.lastname: Frauenhoff
mesh_terms: D000818:Animals; D016227:Benzoquinones / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D008956:Models, Chemical; D010084:Oxidation-Reduction; D011485:Protein Binding; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*
chemical_list: D016227:Benzoquinones; C004532:quinone; D012259:Ribonuclease, Pancreatic