Quantification of protein thiols using ThioGlo 1 fluorescent derivatives and HPLC separation

Abstract

A method for quantification of total soluble protein-derived thiols in beer was developed based on the formation of fluorescent adducts with the maleimide compound ThioGlo1. The problem of interference from fluorescent adducts of sulfite and ThioGlo1 was solved by HPLC separation of the adducts followed by fluorescence detection. Using standard addition of GSH, a detection limit of 0.028 μM thiols was achieved. The application and validation of the method was demonstrated for beers with different color intensities, and the application range is in principle for any biological system containing thiols. However, the quantification of cysteine was complicated by a lower fluorescence response of its ThioGlo1 adducts. Based on the studies of the responses of a series of cysteine-derived thiols and (1)H NMR studies of the structures of ThioGlo1 adducts with GSH and cysteine, it was concluded that thiols with a neighboring free amino group yield ThioGlo1 adducts with a reduced fluorescence intensity.