Analysis of biological thiols: Quantitative determination of thiols at the picomole level based upon derivatization with monobromobimanes and separation by cation-exchange chromatography

Robert C. Fahey, Gerald L. Newton, Randel Dorian, Edward M. Kosower

Research output: Contribution to journalArticlepeer-review

Abstract

A method for quantitative determination of biological thiols is presented. Thiols are converted to fluorescent derivatives by reaction with monobromobimane or monobromotrimethylammoniobimane. The derivatives are separated by ion-exchange chromatography and detected by fluorometry. Thiols that can be separated and quantitated by combined use of mBBr and qBBr derivatives include N-acetylcysteine, coenzyme A, coenzyme M, cysteine, cysteamine, cysteinylglycine, ergothioneine, ethanethiol, glutathione, γ-glutamylcysteine, homocysteine, 2-mercaptoethanol, methanethiol, pantetheine, 4′-phosphopantetheine, thiosulfate, thiouracil, and the mono- and diderivatives of dithiothreitol. Most thiols can be detected at the 1-pmol level and quantitated when present at the 10-pmol or higher level. Some, such as ergothioneine, exhibited low fluorescent yields and can be measured only at levels an order of magnitude higher. The method was applied to human red blood cell where the main thiols were found to be glutathione (2.4 mm) and ergothioneine (120 μm), in accord with earlier reports.

Original languageEnglish
Pages (from-to)357-365
Number of pages9
JournalAnalytical Biochemistry
Volume111
Issue number2
DOIs
StatePublished - 1 Mar 1981

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