Antioxidant Capacity of Free and Peptide Tryptophan Residues Determined by the ORAC (Oxygen Radical Absorbance Capacity) Assay Is Modulated by Radical-Radical Reactions and Oxidation Products.

The ORAC (Oxygen Radical Absorbance Capacity) assay is commonly employed for determining the antioxidant capacity of bioactive peptides. To gain insights into the meaning of this index for peptides containing a single Trp, we studied the consumption of this residue and fluorescein (FLH, the probe of ORAC method), induced by radicals generated by AAPH (2,2'-Azo-bis(2-amidinopropane) dihydrochloride) thermolysis. ORAC values were rationalized from kinetics and computational calculations of bond dissociation energies (BDE) of the N-H bond (indole ring of Trp). Free Trp, di- and tri- peptides, and three larger peptides were studied. Solutions containing 70 nM FLH, 1-5 µM free Trp or peptides, and 10 mM AAPH were incubated at 37 °C in phosphate buffer. Kinetic studies showed that FLH minimally affected Trp consumption. However, a clear protection of FLH, characterized by pseudo-lag times, was evidenced, reflecting radical-radical reactions and FLH repairing. Peptides showed similar ORAC values (~1.9-2.8 Trolox equivalents), while BDE varied between 91.9 and 103.5 kcal. These results, added to the protection of FLH observed after total consumption of Trp, indicate a lack of discrimination of the assay for the chemical structure of peptides and the contribution of oxidation products to the index.

Quantification of carbonate radical formation by the bicarbonate-dependent peroxidase activity of superoxide dismutase 1 using pyrogallol red bleaching.

Carbonate radicals (CO3-) are generated by the bicarbonate-dependent peroxidase activity of cytosolic superoxide dismutase (Cu,Zn-SOD, SOD-1). The present work explored the use of bleaching of pyrogallol red (PGR) dye to quantify the rate of CO3- formation from bovine and human SOD-1 (bSOD-1 and hSOD-1, respectively). This approach was compared to previously reported methods using electron paramagnetic resonance spin trapping with DMPO, and the oxidation of ABTS (2,2-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid). The kinetics of PGR consumption elicited by CO3- was followed by visible spectrophotometry. Solutions containing PGR (5-200 µM), SOD-1 (0.3-3 µM), H2O2 (2 mM) in bicarbonate buffer (200 mM, pH 7.4) showed a rapid loss of the PGR absorption band centered at 540 nm. The initial consumption rate (Ri) gave values independent of the initial PGR concentration allowing an estimate to be made of the rate of CO3- release of 24.6 ±â€¯4.3 µM min-1 for 3 µM bSOD-1. Both bSOD-1 and hSOD-1 showed a similar peroxidase activity, with enzymatic inactivation occurring over a period of 20 min. The single Trp residue (Trp32) present in hSOD-1 was rapidly consumed (initial consumption rate 1.2 ±â€¯0.1 µM min-1) with this occurring more rapidly than hSOD-1 inactivation, suggesting that these processes are not directly related. Added free Trp was rapidly oxidized in competition with PGR. These data indicate that PGR reacts rapidly and efficiently with CO3- resulting from the peroxidase activity of SOD-1, and that PGR-bleaching is a simple, fast and cheap method to quantify CO3- release from bSOD-1 and hSOD-1 peroxidase activity.

Cytoprotective Mechanisms Mediated by Polyphenols from Chilean Native Berries against Free Radical-Induced Damage on AGS Cells.

The prevalence of cytoprotective mechanisms induced by polyphenols such as activation of intracellular antioxidant responses (ICM) and direct free radical scavenging was investigated in native Chilean species of strawberries, raspberries, and currants. Human gastric epithelial cells were co- and preincubated with polyphenolic-enriched extracts (PEEs) from Chilean raspberries (Rubus geoides), strawberries (Fragaria chiloensis ssp. chiloensis f. chiloensis), and currants (Ribes magellanicum) and challenged with peroxyl and hydroxyl radicals. Cellular protection was determined in terms of cell viability, glyoxalase I and glutathione s-transferases activities, and carboxymethyl lysine (CML) and malondialdehyde levels. Our results indicate that cytoprotection induced by ICM was the prevalent mechanism for Rubus geoides and F. chiloensis. This agreed with increased levels of glyoxalase I and glutathione S-transferase activities in cells preincubated with PEEs. ORAC index indicated that F. chiloensis was the most efficient peroxyl radical scavenger. Moreover, ICM mediated by F. chiloensis was effective in protecting cells from CML accumulation in contrast to the protective effects induced by free radical scavenging. Our results indicate that although both polyphenol-mediated mechanisms can exert protective effects, ICM was the most prevalent in AGS cells. These results suggest a potential use of these native berries as functional food.