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Oxidation of polyphenols to quinones serves as an antioxidative mechanism, but the resulting quinones may induce damage to proteins as they react through a Michael addition with nucleophilic groups, such as thiols and amines to give protein adducts. In this study, rate constants for the reaction of 4-methylbenzoquinone (4MBQ) with proteins, thiol and amine compounds were determined under pseudo first-order conditions by UV-vis stopped-flow spectrophotometry. The chemical structures of the adducts were identified by LC-ESI-MS/MS. Proteins with free thiols were rapidly modified by 4MBQ with apparent second order rate constants, k2 of (3.1±0.2)×10(4)M(-1)s(-1) for bovine serum albumin (BSA) and (4.8±0.2)×10(3)M(-1)s(-1) for human serum albumin at pH 7.0. These values are at least 12-fold greater than that for α-lactalbumin (4.0±0.2)×10(2)M(-1)s(-1), which does not contain any free thiols. Reaction of Cys-34 of BSA with N-ethylmaleimide reduced the thiol concentration by ~59%, which resulted in a decrease in k2 by a similar percentage, consistent with rapid adduction at Cys-34. Reaction of 4MBQ with amines (Gly, Nα-acetyl-l-Lys, Nε-acetyl-l-Lys and l-Lys) and the guanidine group of Nα-acetyl-l-Arg was at least 5×10(5) slower than with low-molecular-mass thiols (l-Cys, Nα-acetyl-l-Cys, glutathione). The thiol-quinone interactions formed colorless thiol-phenol products via an intermediate adduct, while the amine-quinone interactions generated colored amine-quinone products that require oxygen involvement. These data provide strong evidence for rapid modification of protein thiols by quinone species which may be of considerable significance for biological and food systems., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The radical-mediated reactions occurring during the early stages of beer storage were studied by following the rate of oxygen consumption, radical formation as detected by electron spin resonance spectroscopy, and concentrations of the antioxidant compounds sulfite and thiols. Addition of either Fe(III) or Fe(II) had similar effects, indicating that a fast redox equilibrium is obtained between the two species in beer. Addition of iron in combination with hydrogen peroxide gave the most pronounced levels of oxidation due to a direct initiation of ethanol oxidation through generation of hydroxyl radicals by the Fenton reaction. The concentration of sulfite decreased more than the thiol concentration, suggesting that thiols play a secondary role as antioxidants by mainly quenching 1-hydroxyethyl radicals that are intermediates in the oxidation of ethanol. Increasing the temperature had a minor effect on the rate of oxygen consumption.

The dose-dependent effects of green tea extract (100, 500, or 1500ppm) on the textural and oxidative stability of meat emulsions were investigated, and compared to a control meat emulsion without extract. All levels of green tea extract inhibited formation of TBARS as a measure for lipid oxidation. Overall protein thiol oxidation and myosin heavy chain (MHC) cross-linking were inhibited by 100ppm green tea extract without jeopardizing the textural stability, while increasing concentrations of extract resulted in reduced thiol concentration and elevated levels of non-reducible protein modifications. Addition of 1500ppm green tea extract was found to modify MHC as evaluated by SDS-PAGE combining both protein staining and specific thiol staining, indicating that protein modifications generated through reactions of green tea phenolic compounds with protein thiols, disrupted the meat emulsion properties leading to reduced water holding capacity and textural stability. Hence, a low dose of green tea extract preserves both the textural and the oxidative stability of the meat proteins., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The antioxidant effects of dried citrus pulp on proteins in lamb meat, when used as a replacement of concentrate in the feed, was studied using meat from 26 male Comisana lambs. The lambs of age 90 days had been grouped randomly to receive one of the three dietary treatments: (1) commercial concentrate with 60% barley (Control, n=8), (2) concentrate with 35% barley and 24% citrus pulp (Cp24, n=9), or (3) concentrate with 23% barley and 35% citrus pulp (Cp35, n=9). Slices from the longissimus thoracis et lomborum muscle were packed aerobically and stored for up to 6days at 4°C in the dark. The citrus pulp groups, Cp24 and Cp35, significantly decreased protein radicals and carbonyls, and preserved more thiols within six days of storage compared to the Control group. The citrus pulp groups significantly slowed down the rate of protein oxidation, indicating that dietary citrus pulp reduced oxidative changes in meat proteins., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The scavenging activity of extracts of green tea (GTE), white grape (WGE), and rosemary (RE), all plant material with high phenolic content, and of the phenolic compounds 4-methylcatechol (4-MC), (+)-catechin, and carnosic acid toward long-lived myosin radicals generated by reaction with H2O2-activated myoglobin at room temperature (pH 7.5, I=1.0) was investigated by freeze-quench ESR spectroscopy. Myosin radicals were generated by incubating 16 μM myosin, 800 μM metmyoglobin, and 800 μM H2O2 for 10 min, and the phenolic extracts were subsequently added (1% (w/w) phenolic compounds relative to myosin). GTE was able to scavenge myosin radicals and reduce the radical intensity by 65%. Furthermore, a low concentration of 4-MC (33 μM) was found to increase the radical concentration when added to the myosin radicals, whereas a higher concentration of 4-MC and catechin (330 μM) was found to scavenge myosin radicals and reduce the overall radical concentration by ∼65%.

The interaction between phenolic compounds and protein thiols was investigated in minced beef with or without 500 ppm 4-methylcatechol (4-MC) that had been stored under oxygen or argon for 7 days (4 °C). Myofibrillar protein isolates were extracted, and the oxidative stability evaluated by the protein radical intensity measured by ESR spectroscopy was found to be improved by 4-MC as well as by storage without oxygen. Significant loss of thiols was found in samples stored under oxygen compared to argon, whereas an additional loss was found in samples with added 4-MC stored under oxygen. In beef with added 4-MC, LC-MS analysis showed formation of thiol-quinone adducts, which may explain the observed additional loss of thiols. Although storage without oxygen inhibited protein cross-link formation as evaluated by SDS-PAGE, both in presence and in the absence of 4-MC, no inhibitory effect of 4-MC was found on the formation of protein disulfide cross-links in beef stored under oxygen.

A combinatorial chemistry approach was employed for the design and systematic synthesis of antioxidant-emulsifier bioconjugates to improve antioxidant activity in the interface between oil and water. A combinatorial library of 12 bioconjugates was synthesized from the phenolic antioxidants Trolox (a water-soluble alpha-tocopherol analogue), dihydroferulic acid, dihydrocaffeic acid, and gallic acid in combination with serine ethyl ester, serine lauryl ester, and lauroyl serine by esterification of the serine side chain or amidation, respectively. The bioconjugates were characterized by NMR and mass spectrometry, and each was tested for antioxidant activity by measuring the radical scavenging rate of 2,2-diphenyl-1-picrylhydrazyl (DPPH (*)) in methanol, the radical scavenging rate of DPPH (*) in a heterogeneous solvent system, the rate of oxygen consumption in an oil-in-water emulsion with metmyoglobin initiated oxidation, and the lag phase for diene formation in unilamellar liposomes with free radical initiation in the aqueous phase; each case was compared to the antioxidant activity of the parent antioxidant. In general, the conjugates with longer chain derivatives exhibited improved antioxidative activity in heterogeneous systems, whereas no improvement was observed in homogeneous solution. The rate of oxygen consumption in oil-in-water emulsion was found to decrease with increasing octanol/water partition coefficient, which is suggested to correspond to a saturation of the water/oil interface with antioxidant bioconjugate to approach maximal protection.

The antioxidative effect of purified carnosine (i.e., separated from the common contaminant hydrazine) has been evaluated in two systems: (i) Carnosine was found to possess poor reducing properties toward the prooxidant ferrylmyoglobin; at pH approximately 5 the presence of carnosine did not increase the rate of reduction of MbFe(IV)=O compared to autoreduction, whereas at pH 7.4 the rate constant for reduction by carnosine was 0.010 +/- 0.002 M(-1).s(-1) (I = 0.16; 25.0 degrees C). (ii) In cooked pork patties prepared from meat (longissimus dorsi and masseter) with purified or nonpurified carnosine added, the effect of purified carnosine was insignificant when compared to control patties, whereas patties with carnosine contaminated with hydrazine had a lower oxidation level than patties with purified carnosine. Carnosine is concluded not to deactivate the prooxidant ferrylmyoglobin and not to have any antioxidative effect in cooked pork.