A modification of the ABTS• decolorization assay for plate readers is presented. In our modification, 200 µL of ABTS solution of absorbance 1.0 at 734 nm was added with an antioxidant and decreased absorbance resulted. For comparison of antioxidant activities in the kinetic assay of absorbance decrease, concentration dependence of absorbance decrease and of area under curve are recommended. “Fast” and “slow” antioxidants were distinguished: while the reactions of “fast” antioxidants ABTS• were completed within seconds, the reactions of “slow” antioxidants were not finished after 6 min. We recommend reaction time of 60 min for assays of such antioxidants, blood plasma and plant extracts. Sub-additive interactions between some antioxidants (ascorbate and Trolox, hispidulin and Trolox, and glutathione and ascorbate) were found in the ABTS• decolorization; possible reasons for such interactions are discussed.
There are conflicting reports on the antioxidant activity of hispidulin. Antioxidant activity of hispidulin was evaluated using assays of ABTS • reduction, ferric ion reducing antioxidant power (FRAP) assay, DPPH reduction assay, and protection of erythrocyte membranes against lipid peroxidation and protein thiol oxidation. ABTS • reduction assay pointed to the involvement of all three phenol groups of hispidulin in ABTS • reduction. The reactivity of hispidulin in the FRAP assay and DPPH reduction assay was low (0.09 and 0.019 of the reactivity of Trolox). However, hispidulin was effective in protection against erythrocyte membrane lipid peroxidation and highly effective in protection against erythrocyte membrane protein thiol group oxidation (more effective than Trolox). These results point to the necessity of caution in extrapolating the antioxidant activity evaluated in simple cell-free systems on more complex systems.
The reaction of the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) free radical (ABTS●) with proteins (bovine serum albumin, blood plasma, egg white, erythrocyte membranes, and Bacto Peptone) leads not only to a reduction of ABTS● but also to the appearance of a purple color (absorption maximum at 550–560 nm). The aim of this study was to characterize the formation and explain the nature of the product responsible for the appearance of this color. The purple color co-precipitated with protein, and was diminished by reducing agents. A similar color was generated by tyrosine upon reaction with ABTS●. The most feasible explanation for the color formation is the addiction of ABTS● to proteins’ tyrosine residues. The product formation was decreased by nitration of the bovine serum albumin (BSA) tyrosine residues. The formation of the purple product of tyrosine was optimal at pH 6.5. A decrease in pH induced a bathochromic shift of the spectra of the product. The product was not a free radical, as demonstrated by electrom paramagnetic resonance (EPR) spectroscopy. Another byproduct of the reaction of ABTS● with tyrosine and proteins was dityrosine. These byproducts can contribute to the non-stoichiometry of the antioxidant assays with ABTS●. The formation of the purple ABTS adduct may be a useful index of radical addition reactions of protein tyrosine residues.
It has been estimated and demonstrated that the antioxidant capacity of proteins is increased as a result of digestion in the gastrointestinal tract, which can be contributed by denaturation and digestion. This study aimed to evaluate the effect of denaturation and proteolytic digestion on the antioxidant activity of bovine serum albumin (BSA) and chicken egg white proteins in model systems. Denaturation with an anionic detergent (sodium dodecyl sulfate) and digestion with papain and trypsin increased the antioxidant activity/capacity of the proteins, apparently due to the increased exposure of amino acid residues responsible for the antioxidant activity of proteins (tyrosine, tryptophan, cysteine, histidine, arginine, and cystine in the ABTS● decolorization assay; cysteine, tryptophan, tyrosine, and cystine in the FRAP assay). As the increase in the protein antioxidant activity/capacity was limited in extent, it does not invalidate the use of the antioxidant capacity of proteins to be consumed as a rough measure of their antioxidant capacity after modifications in the gastrointestinal tract.
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