Reduction of sulfenic acids by ascorbate in proteins, connecting thiol-dependent to alternative redox pathways

Sulfenic acids are the primary product of thiol oxidation by hydrogen peroxide and other oxidants. Several aspects of sulfenic acid formation through thiol oxidation were established recently. In contrast, the reduction of sulfenic acids is still scarcely investigated. Here, we characterized the kinetics of the reduction of sulfenic acids by ascorbate in several proteins. Initially, we described the crystal structure of our model protein (Tsa2-C170S). There are other Tsa2 structures in distinct redox states in public databases and all of them are decamers, with the peroxidatic cysteine very accessible to reductants, convenient features to investigate kinetics. We determined that the reaction between Tsa2-C170S-Cys-SOH and ascorbate proceeded with a rate constant of 1.40 ± 0.08 × 103 M−1 s−1 through a competition assay developed here, employing 2,6–dichlorophenol-indophenol (DCPIP). A series of peroxiredoxin enzymes (Prx6 sub family) were also analyzed by this competition assay and we observed that the reduction of sulfenic acids by ascorbate was in the 0.4–2.2 × 103 M−1 s−1 range. We also evaluated the same reaction on glyceraldehyde 3-phosphate dehydrogenase and papain, as the reduction of their sulfenic acids by ascorbate were reported previously. Once again, the rate constants are in the 0.4–2.2 × 103 M−1 s−1 range. We also analyzed the reduction of Tsa2-C170S-SOH by ascorbate by a second, independent method, following hydrogen peroxide reduction through a specific electrode (ISO-HPO-2, World Precision Instruments) and employing a bi-substrate, steady state approach. The was 7.4 ± 0.07 × 103 M−1 s−1, which was in the same order of magnitude as the value obtained by the DCPIP competition assay. In conclusion, our data indicates that reduction of sulfenic acid in various proteins proceed at moderate rate and probably this reaction is more relevant in biological systems where ascorbate concentrations are high

ANTIOXIDANT ACTIVITY IN RELATION TO THE SIZE OF POD IN DIFFERENT SNAP BEAN GENOTYPES

INTRODUCTION: The snap bean (Phaseolus vulgaris L.) is a legume that is widely consumed on the world scenario due to its biochemical composition and nutritional quality (Furlan et al., 2016). Besides the nutritional compounds, the snap bean presents antioxidants properties, inhibiting or retarding the oxidative damage, which avoids the propagation of oxidative reactions and can prevent diseases caused by free radicals. (Silva et al., 2009). The aim of this study was to evaluated the interaction between the antioxidant activity and pod size of several snap bean genotypes cultivated in the sowing season spring/summer and autumn/winter. MATERIAL AND METHODS: The experiment was conducted in an organic system with protected cultivation in the municipality of Londrina, Paraná, Brazil. The study was conducted in a completely randomized design, in a factorial scheme (8 vs. 2), with four replications, being eigth snap bean genotypes with indeterminate growth pattern (Teresópolis Ag 481, HAV 69, HAV 41, Preferido Ag 482, Macarrão Brasília, Trepador Top Seed, HT 30 e Favorito Ag 480) and tow sowing season: spring/summer of 2014 and autumn/winter of 2015. Each experimental plot was composed by 10 plants spaced at 0.20 m in the line and 1.00 m between lines. Samples of pods were collected from each treatment, being measured: the average pod mass, length and diameter of pods - measured in 10 pods per plant; And antioxidant activity (RUFINO et al., 2007). The data were submitted to analysis of variance by the F test (p \u3c0.05) and compared by the principal component analysis (PCA), using software R (R, 2012)