The determination of free sulfhydryl groups is important
in many aspects of biotechnology, such as measuring the
coupling efficiency of thiol-reactive probes, assaying cysteine-
containing haptens, and assaying reductase/thiol
transferase activity, as well as in various aspects of the
food industry. This is generally achieved colorimetrically
using Ellman’s reagent, although the assay is relatively
insensitive and Ellman’s reagent is unstable. In this
paper, we describe a highly sensitive fluorometric assay
for free sulfhydryl groups based on FRET, which we have
used to develop a sensitive assay for glutathione reductase
activity. The assay exploits the specific increase in fluorescence
intensity that occurs at 520 nm when a probe
containing two molecules of fluorescein linked via a
disulfide group is cleaved by glutathione. The assay is 2
orders of magnitude more sensitive than the commonly
used colorimetric glutathione reductase assay and has a
greater dynamic range.