Determination of thiol proteins using monobromobimane labeling and high-performance liquid chromatographic analysis: application to Escherichia coli thioredoxin.

A highly sensitive and specific assay for Escherichia coli thioredoxin was developed using the thiol-specific reagent monobromobimane. Treatment of dithiothreitol-reduced thioredoxin with an excess of monobromobimane in Tris buffer (pH 8.0, 23 degrees C) for 30 min resulted in the formation of a stable derivative which was quantitated by reverse-phase high-performance liquid chromatography with fluorescence detection providing sensitivity in the low picomole range. This method was applied to the determination of intracellular levels of thioredoxin in E. coli. Cell extracts were heated, treated with dithiothreitol, reacted with monobromobimane, and desalted to give a solution which was analyzable for thioredoxin using the chromatographic procedure.