Analysis of biological thiols: Quantitative determination of thiols at the picomole level based upon derivatization with monobromobimanes and separation by cation-exchange chromatography
✍ Scribed by Robert C. Fahey; Gerald L. Newton; Randel Dorian; Edward M. Kosower
- Publisher
- Elsevier Science
- Year
- 1981
- Tongue
- English
- Weight
- 678 KB
- Volume
- 111
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
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, y-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 IO-pm01 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 PM), in accord with earlier reports. DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); NEM, Nethylmaleimide; CoM, 2-mercaptoethanesulfonic acid.
* The bromobimanes are avilable from Calbiochem-Behring Corporation under the trade names "thiolyte MB" (mBBr) and "thiolyte-monoquat" (qBBr).