Covalent binding of benzoquinone to reduced ribonuclease. Adduct structures and stoichiometry

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 inve...

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Bibliographic Details
Published in:Chemical research in toxicology 1994-03, Vol.7 (2), p.177-184
Main Authors: HANZLIK, R. P, HARRIMAN, S. P, FRAUENHOFF, M. M
Format: Article
Language:English
Subjects:
Animals
Benzoquinones - metabolism
Binding Sites
Biological and medical sciences
Cattle
Chemical and industrial products toxicology. Toxic occupational diseases
Medical sciences
Models, Chemical
Oxidation-Reduction
Protein Binding
Ribonuclease, Pancreatic - metabolism
Toxicology
Various organic compounds
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Summary: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.
ISSN:0893-228X
1520-5010
DOI:10.1021/tx00038a010