Analysis of protein-glutathione mixed disulfides by high performance liquid chromatography

✍ Scribed by Michael J. Meredith

• Publisher: Elsevier Science
• Year: 1983
• Tongue: English
• Weight: 437 KB
• Volume: 131
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(83)90205-1

✦ Synopsis

After precipitation of proteins; serum, hepatocytes, or glutathione-derivatized bovine serum albumin, by perchloric acid, dithiothreitol was used to reduce glutathione-protein mixed disulfides in the ether-washed, resuspended pellet. Following neutralization and Scarboxymethylation of free s&hydra1 groups in the acid soluble fraction by iodoacetic acid, 2,4-dinitrophenyl derivatives of released compounds were produced by addition of ethanolic fluorodinitrobenzene. The 2,4-dinitrophenyl derivative of Scarboxymethylglutathione was measured by high-performance liquid chromatography.

The method was found to be reproducible and limited only by the sensitivity of the ghrtathione analysis (about 10 pmol/sample). Quantitation of protein-bound glutathione was shown to be independent of the ratio of bound to soluble glutathione as well as the protein concentration in the sample. This method was found to produce ghrtathione values identical to those measured after borohydride reduction without the problems of foaming, sample loss, and the need of continuous pH adjustment during reduction.