[20] Assay for thiols based on reactivation of papain

This chapter describes a sensitive spectrophotometric assay for the quantitation of thiol groups that is based on reactivation of papain. The assay involves the reaction of a thiol with an inactive mixed disulfide of papain, resulting in the stoichiometric formation of active papain. The activated papain catalyzes the hydrolysis of a chromogenic substrate that generates an amplified spectrophotometric signal proportional to the initial amount of thiol. This enzyme-amplified assay is about 100-fold more sensitive than Ellman's assay. Amounts of thiol as small as 0.06 nmol have been detected accurately in routine applications of the assay. In the assay, the reaction of a thiol with an excess of papain-S-SCH3 results in the stoichiometric formation of active papain, which can be assayed with a chromogenic substrate, such as N-benzoyl-l-arginine-p-nitroanilide. The assay depends on the rapid and complete thiol–disulfide interchange. The reaction may be slow when the thiol to be assayed has a high pKa (low concentration of the reactive thiolate form) or is sterically less accessible. The reactions between these thiols and papain-S-SCH3 can be accelerated by the addition of a large excess (10- to 100-fold) of a disulfide, such as cystamine. The indirect thiol–disulfide interchange reactions mediated by cystamine result in the stoichiometric formation of active papain, because the overall equilibrium of the reaction between thiols (pKa ∼8–11) and papain-S-SCH3 lies significantly toward the formation of papain-SH (pKa ∼4), even in the presence of a 10- to 100-fold molar excess of cystamine.