Measurement of glutathione-protein mixed disulfides

ORCiD

0000-0001-9010-5970

Document Type

Article

Publication Title

International Journal of Radiation Oncology - Biology - Physics

ISSN

0360-3016

Volume

10

Issue

9

DOI

10.1016/0360-3016(84)90491-7

First Page

1507

Last Page

1510

Publication Date

9-1-1984

Abstract

We have undertaken the development of a sensitive and highly specific assay for the presence of mixed disulfides between protein thiol groups and endogenous thiols. Previous investigations on the concentrations of glutathione (GSH), glutathione disulfide (GSSG) and protein glutathione mixed disulfides (ProSSG) have been of limited usefulness because of the poor specificity of the assays used. Our assay for these forms of glutathione is based on high performance liquid chromatography (HPLC) and is an extension of an earlier method. After perchloric acid precipitation, the protein sample is washed with an organic solvent to fully denature the protein. Treatment with 25 mM dithiothreitol (DTT) in 50 mM N-morpholinopropane sulfonic acid buffer, pH 8.0, reduces disulfides on the protein, and free thiols resulting from this procedure are analyzed by HPLC. We have found up to a 10-fold increase in GSH released from fetal bovine serum (FBS) protein when the protein precipitate is washed with ethanol rather than ether, as earlier suggested. Similar effects have been observed with an as yet unidentified thiol which elutes in our chromatography system with a retention volume similar to cysteine. Future experiments concerning this unidentified thiol are in progress.

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