An immunoblot assay for cysteine oxidation by reactive oxygen species allows detection of novel thioprotective efficacy of black tea extracts.

ABSTRACT

INTRODUCTION: While cysteine thiol groups help to maintain the redox status of many proteins, they can be very susceptible to damaging oxidants. Despite broad interest in their antioxidant properties, whether tea polyphenols protect against protein thiol damage of this kind is unclear. This study sought to develop a simple immunoassay for use in screening tea extracts and other antioxidants for thioprotective efficacy at protein thiol groups. METHODS: Fresh aqueous extracts were prepared from commercially sourced green, white, black and red teas. Traut's reagent (2-iminothiolane) was used to prepare surface-thiolated bovine serum albumin for use as assay substrate in the protein oxidation assay. Oxidative damage was induced during a 15 min incubation with hydrogen peroxide (H2O2) in the presence of tea extracts and reference antioxidants. The substrate protein was then derivatised with dimedone before samples were loaded onto a nitrocellulose membrane housed within a Slot-Blot apparatus. After blocking nonspecific protein binding a commercially available antibody was used to detect dimedone-labelled groups. RESULTS: While the total phenol content of tea extracts typically correlated with their activity in lipid peroxidation and galvinoxyl radical-trapping assays, the former did not fully predict their abilities to suppress H2O2-induced cysteine oxidation, with black tea extracts displaying greater activity than the other teas and an apparent ability to reverse pre-existing cysteine oxidation. Among the model antioxidants tested, quercetin displayed a heightened ability to suppress cysteine oxidation. DISCUSSION: This slot-blot immunoassay is a convenient method that facilitates standardised comparisons between the thioprotective properties of structurally- and constitutively-diverse antioxidants.