An Amplified Assay for Thiols Based on Reactivation of Papain

Abstract

A sensitive spectrophotometric assay has been developed for thiol (sulfhydryl) groups using an inactive di-sulfide derivative of papain (papain-S-SCH3). The thiol-disulfide interchange reaction of a thiol with papain-S-SCH3 results in the stoichiometric formation of active papain (papain-SH). The reactivated papain catalyzes the hydrolysis of a chromogenic substrate, resulting in an amplified spectrophotometric signal proportional to the initial amount of thiol. A variety of thiols, e.g., cysteine, glutathione, penicillamine, cysteine methyl ester, and cysteamine, yield similar linear plots for the activity of papain vs the initial amount of thiol. An unknown concentration of a thiol is measured using a standard plot for the activity of papain vs the amount of thiol, obtained for the same thiol or for a similar thiol. Thiol groups on proteins and thiol groups of high values of pKa(2-mercaptoethanol, 3-mercaptopropanoic acid) can also be assayed using papain-S-SCH3 in the presence of excess cystamine. The assay is about 100-fold more sensitive than that using Ellman′s reagent [5,5′-dithiobis(2-nitrobenzoic acid)]. A 0.4 μM solution of cysteine produces an absorbance change of 0.55 at 410 nm after 30 mm in the assay, compared to a predicted change in absorbance of 0.0054 using Ellman′s assay.