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Evaluation of the micromethod for determination of glutathione using enzymatic cycling and Ellman's reagent

✍ Scribed by Peter Eyer; Dušan Podhradský

Publisher
Elsevier Science
Year
1986
Tongue
English
Weight
838 KB
Volume
153
Category
Article
ISSN
0003-2697

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✦ Synopsis

Evaluation of the kinetic parameters of the various reactions involved in the determination of ghttathione provided the rationale for a modification of the frequently used assay (F. Tietze, 1969, Anal. Biochem. 27, 502-522) whereby the enzymatic reaction is no longer rate limiting. At pH 6.0, the nonenzymatic thiol interchange reaction of reduced glutathione (GSH) with Ellman's reagent becomes rate limiting, and inhibition of ghttathione reductase up to 50% has no influence on the accuracy of the determination. The lower level of sensitivity is lo-" mol ghttathione with a linear response up to 5 X 1 Om9 mol. For determination of ghttathione disulfide, GSH is alkylated by N-ethylmaleimide (NEM), and excess NEM is removed by extraction with ethyl acetate. Since the glutathione adduct is not stable, extracted samples are kept deep-frozen prior to analysis, Using this precaution, less than 0.05% of GSSG was determined in GSH-containing samples which had been previously freed from GSSG. 0 1986 Academic PPX, ILK.

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