Document Detail

77Se NMR characterization of 77Se-labeled ovine erythrocyte glutathione peroxidase.
MedLine Citation:
PMID:  2002027     Owner:  NLM     Status:  MEDLINE    
Lambs, maintained on a selenium-deficient diet supplemented with 94 atom % Na2 27SeO3, have been used as a source of 77Se-enriched erythrocyte glutathione peroxidase. After 5 months on this diet, the percentage of selenium in the enzyme derived from the supplement had reached 88%. From each monthly bleeding of two sheep, approximately 20 mg of 77Se-enriched glutathione peroxidase could be isolated in pure form. Although attempts to observe 77Se NMR signals from the native enzyme labeled with 6,6'-[77Se]diselenobis-(3-nitrobenzoic acid) failed, due to the low solubility of the enzyme, two 77Se resonances were observed after unfolding the enzyme with 8 M urea and reaction with iodoacetamide. These resonances, at 195 and 377 ppm, were from the selenoether alkylamide derivative and from protein cross-linked selenide sulfide species, respectively. Relaxation time measurements on the selenoether at 4.7 and 9.4 teslas enabled an estimate of the chemical shift anisotropy to be made. A value of less than or equal to 262 ppm was determined. Reduction of the denatured selenide sulfide species with dithiothreitol gave an observable 77Se resonance from the Se- moiety at pH 8 and from SeH at pH 4.2. The chemical form of the selenocysteine residue in the resting state enzyme most consistent with formation of the acetamide derivative and the selenide sulfide is Se- or SeH. From the magnitudes of the estimated chemical shift anisotropies, it is predicted that direct observation of selenium in the native enzyme will be feasible if the enzyme concentration can be increased to 0.25 mM tetrameric glutathione peroxidase.
P Gettins; B C Crews
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  266     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  1991 Mar 
Date Detail:
Created Date:  1991-04-17     Completed Date:  1991-04-17     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  4804-9     Citation Subset:  IM    
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
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MeSH Terms
Erythrocytes / enzymology*
Glutathione Peroxidase / chemistry*
Magnetic Resonance Spectroscopy
Grant Support
Reg. No./Substance:
0/Isotopes; 3483-12-3/Dithiothreitol; 7782-49-2/Selenium; EC Peroxidase

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