Gerald L. Newton, R. C. Fahey
1995
Citations
3
Influential Citations
111
Citations
Quality indicators
Journal
Methods in enzymology
Abstract
Publisher Summary This chapter discusses the determination of biothiols by bromobimane labeling and high-performance liquid chromatography (HPLC). Most thiols undergo rapid autoxidation when exposed to air at neutral or basic pH, a process that is catalyzed by heavy metals of the type found in most cells. A primary consideration in preparing and analyzing samples for thiols is to prevent their oxidative loss during the sample preparation and analysis. This can be accomplished by alkylation of the thiol group near neutral pH with the bromobimanes. The chapter focuses on monobromobimane (mBBr), an uncharged reagent that readily penetrates cells, 1-4 and 4- p -sulfobenzoyloxymethyl-6-bromomethy-1-3,7-dimethyl-1,5-diazabicyclo [3.3.0] octa-3,6-diene-2,8-dione (SBBr), an anionic reagent that does not enter cells. Many biological thiols are relatively small and ionic, which makes them easy to separate from more hydrophobic thiols but difficult to separate from each other using reversed-phase HPLC. The bimane derivatives of these ionic thiols are more hydrophobic than the thiol itself and are readily resolved by HPLC analysis on silica-based reversed-phase columns. The highly selective, rapid reactivity of bromobimanes toward thiols, the stability and fluorescence yield of the thiol derivatives, the ease of separation of the derivatives by reversed-phase HPLC, and the availability of both cell-penetrating and nonpenetrating forms make the use bromobimanes an extremely powerful approach to the analysis of low-molecular-weight biothiols.