Reduction Of Ferrylmyoglobin Research Articles

Reduction of ferrylmyoglobin by cysteine as affected by pH

Herein we report the kinetics and mechanism by which hypervalent heme-iron species formed in the gut may be deactivated by thiols like cysteine and glutathione.

Reduction of Ferrylmyoglobin by Theanine and Green Tea Catechins. Importance of Specific Acid Catalysis

Reduction of the hypervalent heme pigment ferrylmyoglobin by green tea catechins in aqueous solution of pH = 7.5 was investigated by stopped-flow spectroscopy. Reduction by the gallic acid esters epigallocatechin gallate (EGCG, k2 = 1460 L mol(-1) s(-1), 25.0 °C, 0.16 ionic strength) and epicatechin gallate (ECG, 1410 L mol(-1) s(-1)) was found faster than for epicatechin (EC, 300 L mol(-1) s(-1)) and epigallocatechin (EGC, 200 L mol(-1) s(-1)), even though the gallate ion (G, 330 L mol(-1) s(-1)) is similar in rate to EC. The rate for reduction by EC, EGC, ECG, EGCG, and G shows no correlation with their oxidation potentials or phenolic hydrogen-oxygen bond dissociation energy, but with the pKa of the most acidic phenol group. Theanine, with an acidity similar to that of EC, reduces ferrylmyoglobin with a similar rate (200 L mol(-1) s(-1)), in support of general acid catalysis with an initial proton transfer prior to electron transfer.

Reduction of Ferrylmyoglobin by Hydrogen Sulfide. Kinetics in Relation to Meat Greening

The hypervalent meat pigment ferrylmyoglobin, MbFe(IV)═O, characteristic for oxidatively stressed meat and known to initiate protein cross-linking, was found to be reduced by hydrogen sulfide to yield sulfmyoglobin. Horse heart myoglobin, void of cysteine, was used to avoid possible interference from protein thiols. For aqueous solution, the reactions were found to be second-order, and an apparent acid catalysis could be quantitatively accounted for in terms of a fast reaction between protonated ferrylmyoglobin, MbFe(IV)═O,H(+), and hydrogen sulfide, H2S (k2 = (2.5 ± 0.1) × 10(6) L mol(-1) s(-1) for 25.0 °C, ionic strengh 0.067, dominating for pH < 4), and a slow reaction between MbFe(IV)═O and HS(-) (k2 = (1.0 ± 0.7) × 10(4) L mol(-1) s(-1) for 25.0 °C, ionic strengh 0.067, dominating for pH > 7). For meat pH, a reaction via the transition state {MbFe(IV)═O···H···HS}([symbol: see text]) contributed significantly, and this reaction appeared almost independent of temperature with an apparent energy of activation of 2.1 ± 0.7 kJ mol(-1) at pH 7.4, as a result of compensation among activation energies and temperature influence on pKa values explaining low temperature greening of meat.

Inhibition of the metmyoglobin-induced peroxidation of linoleic acid by dietary antioxidants: Action in the aqueous vs. lipid phase

The gastric digestion of food containing oxidizable lipids and iron catalysts for peroxide decomposition such as (met)myoglobin from muscle meat can be accompanied by an extensive formation of potentially toxic lipid hydroperoxides. An early protective action by dietary antioxidants in the gastro-intestinal tract is plausible, especially for poorly bioavailable antioxidants such as polyphenols. Hence, the ability of antioxidants to inhibit lipid peroxidation initiated by dietary iron in mildly acidic emulsions is a valuable and general model. In this work, the ability of some ubiquitous dietary antioxidants representative of the main antioxidant classes (α-tocopherol, the flavonol quercetin, β-carotene) to inhibit the metmyoglobin-induced peroxidation of linoleic acid is investigated by UV–visible spectroscopy and HPLC in mildly acidic emulsions. The phenolic antioxidants quercetin and α-tocopherol come up as the most efficient peroxidation inhibitors. Inhibition by quercetin essentially proceeds in the aqueous phase via a fast reduction of an unidentified activated iron species (with a partially degraded heme) produced by reaction of metmyoglobin with the lipid hydroperoxides. This reaction is faster by, at least, a factor 40 than the reduction of ferrylmyoglobin (independently prepared by reacting metmyoglobin with hydrogen peroxide) by quercetin. By contrast, α-tocopherol mainly acts in the lipid phase by reducing the propagating lipid peroxyl radicals. The poorer inhibition afforded by β-carotene may be related to both its slower reaction with the lipid peroxyl radicals and its competitive degradation by autoxidation and/or photo-oxidation.

Reduction of ferrylmyoglobin by the spin trap N-tert-butyl-α-phenylnitrone (PBN) in aqueous solution and during freezing

The hypervalent muscle pigment ferrylmyoglobin, formed by activation of metmyoglobin by hydrogen peroxide, was found to be reduced in a second-order reaction by N-tert-butyl-α-phenylnitrone (PBN, often used as a spin trap). In acidic aqueous solution at ambient temperature, the reduction is relatively slow (δH‡ = 65 ± 2 kJ · mol-1 and δS‡ = -54 ± 7 J · mol-1. K-1 for pH = 5.6), but phase transitions during freezing of the buffered solutions accelerates the reaction between ferrylmyoglobin and PBN. In these heterogenous systems at low temperature (but not when ice-formation was inhibited by glycerol), a PBN-derived radical intermediate was detected by ESR-spectroscopy, identified as a nitroxyl radical by a parallel nitrogen hyperfine coupling constant of 31.8 G, and from microwave power saturation behavior concluded not to be located in the heme-cleft of the protein. The acceleration of the reaction is most likely caused by a lowering of the pH during the freezing of the buffered solutions whereby ferrylmyoglobin becomes more oxidizing.

Factors Influencing the Antioxidant Activity Determined by the ABTS•+ Radical Cation Assay

This study introduces a simple direct antioxidant assay, based on the reduction of the ABTS.+ radical cation, and compares it with the myoglobin/ABTS.+ assay. The methods give closely similar results, establishing that the antioxidants studied to date in the latter assay act by scavenging the ABTS.+ radical cation and not by inhibiting its formation through reduction of ferryl myoglobin or reaction with H2O2.

The reduction of ferryl myoglobin by phenols

The reduction of ferryl myoglobin by phenols

Reduction of ferrylmyoglobin by beta-lactoglobulin.

Reduction of iron (IV) in ferrylmyoglobin in the presence of beta-lactoglobulin in aqueous solution is the result of two parallel reactions: (i) a so-called autoreduction, and (ii) reduction by beta-lactoglobulin in a second-order-reaction resulting in bityrosine formation in beta- lactoglobulin. In the pH-region investigated (5.4-7.4), the rate of reduction increased for both reactions with decreasing pH. The second order-reaction had for non-denatured beta-lactoglobulin the activation parameters: delta H* = 45 kJ.mol-1 and delta S not equal to = -93 J.mol-1.K-1 at pH = 7.0 and ionic strength 0.16 (NaCl). Reduction of ferrylmyoglobin by beta-lactoglobulin denatured by heat (86 degrees C for 3 min) or by hydrostatic pressure (300 MPa for 15 min) resulted in formation of higher molecular weight species as detected by size-exclusion chromatography and by SDS-PAGE. No molecular weight changes were observed for reduction of ferrylmyoglobin by native beta-lactoglobulin. Detection of bityrosine in the native beta-lactoglobulin fraction after oxidation with ferrylmyoglobin indicated intra-molecular bityrosine formation. In heat-denatured beta-lactoglobulin bityrosine formation could be of intra-molecular and/or of inter-molecular origin, the latter being confirmed by size-exclusion chromatography.

Reduction of ferrylmyoglobin by dietary phenolic acid derivatives of cinnamic acid

The reaction of dietary phenolic acid derivatives of cinnamic acid with high valence states of horse myoglobin was studied. When metmyoglobin was oxidized by hydrogen peroxide (H 2O 2) in the presence of the phenolic acids, ferrylmyoglobin was reduced to metmyoglobin. However, addition of the phenolic acids to a ferrylmyoglobin solution resulted in a modified metmyoglobin spectrum characterized mainly by a blue shift of the 631 nm peak and a new isosbestic point at 613 nm suggesting an irreversible modification of the hemeprotein. The efficiency and the kinetic profile of ferrylmyoglobin reduction were dependent on both the concentration and the structure of the phenolic acid. Electron-donating substituent groups in the phenol ring increased the efficiency of ferrylmyoglobin reduction whereas electron-withdrawing groups decreased it. The phenolic acids exhibiting a catechol structure were the most efficient in reducing ferrylmyoglobin. Caffeic and chlorogenic acids react faster than trolox, and caffeic acid faster than ascorbate. During the electron-transfer reactions, the phenolic acids were oxidized to quinoid forms and, in some cases, to polymer products as indicated by comparison of the ultraviolet (UV) spectra obtained in the presence of metmyoglobin/H 2O 2 with those recorded after oxidation of phenolic acids by horseradish peroxidase/H 2O 2 and catechol oxidase. The results suggest that dietary phenolic derivatives of cinnamic acid may counteract deleterious oxidations initiated by ferrylmyoglobin.

Reduction of ferrylmyoglobin and ferrylhemoglobin by nitric oxide: a protective mechanism against ferryl hemoprotein-induced oxidations.

The reactions of metmyoglobin (metMb) and methemoglobin (metHb), oxidized to their respective oxoferryl free radical species (.Mb-FeIV = O/.Hb-4FeIV = O) by tert-butyl hydroperoxide (t-BuOOH), with nitric oxide (NO.) were studied by a combination of optical, electron spin resonance (ESR), ionspray mass (MS), fluorescence, and chemiluminescence spectrometries to gain insight into the mechanism by which NO. protects against oxidative injury produced by .Mb-FeIV = O/.Hb-4FeIV = O. Oxidation of metMb/metHb by t-BuOOH in a nitrogen atmosphere proceeded via the formation of two protein electrophilic centers, which were heme oxoferryl and the apoprotein radical centered at tyrosine (for the .Mb-FeIV = O form, the g value was calculated to be 2.0057), and was accompanied by the formation of t-BuOOH-derived tert-butyl(per)oxyl radicals. We hypothesized that NO. may reduce both oxoferryl and apoprotein free radical electrophilic centers of .Mb-FeIV = O/.Hb-4FeIV = O and eliminate tert-butyl(per)oxyl radicals, thus protecting against oxidative damage. We found that NO. reduced .Mb-FeIV = O/.Hb-4FeIV = O to their respective ferric (met) forms and prevented the following: (i) oxidation of cis-parinaric acid (PnA) in liposomes, (ii) oxidation of luminol, and (iii) formation of the tert-butyl(per)oxyl adduct with the spin trap DMPO. NO. eliminated the signals of tyrosyl radical detected by ESR and oxoferryl detected by MS in the reaction of t-BuOOH with metMb. As evidenced by MS of apomyoglobin, this effect was due to the two-electron reduction of .Mb-FeIV = O by NO. at the oxoferryl center rather than to nitrosylation of the tyrosine residues. Results of our in vitro experiments suggest that NO. exhibits a potent, targetable antioxidant effect against oxidative damage produced by oxoferryl Mb/Hb.

Reduction of ferrylmyoglobin in rat diaphragm

The oxidation of myoglobin was monitored by transmission spectroscopy in isolated, superfused preparations of rat diaphragms. In its deoxygenated form, during anoxia, myoglobin was oxidized by adding hydrogen peroxide (1.0 mM) to its ferryl form (FeIV). On the other hand, peroxide-induced formation of ferrylmyoglobin was not observed when the perfusate contained oxygen. Ferrylmyoglobin was visualized after its derivatization with Na2S to form sulfmyoglobin. Depending on the time of addition, ascorbate (4.0 mM) or ergothioneine (2.0 mM) either prevented the formation of or dissipated ferrylmyoglobin. These agents are known to be reductants of this hypervalent form of myoglobin. In addition to providing the first demonstration of ferrylmyoglobin in skeletal muscle, these observations are consistent with the concept that oxidation of myoglobin to hypervalent states might be an important event in the initiation of muscle damage associated with anoxia and reoxygenation. The rapid reduction of myoglobin would prevent peroxidatic alterations of essential cellular constituents by ferrylmyoglobin.

The reduction of ferryl myoglobin by ergothioneine: A novel function for ergothioneine

In this paper, we demonstrate that ergothioneine (ES), a naturally occurring thiolhistidine, reduces ferrylmyoglobin (Mb IV) to Mb III when the former (ferryl species) is produced by exposing either deoxy Mb II or Mb III to H 2O 2. The reduction of Mb IV to Mb III by ES yields the disulflde of ES which the addition of GSH promptly reduces back to ES. The addition of ES (100 μ m) in the perfusion buffer of Langendorff rat heart preparations exposed to a brief period of ischemia prevents the myocardial damage (lactate dehydrogenase release) which accompanies reperfusion. The results of these experiments support a view that ES and its redox couple GSH might function in a Mb redox cycle.

Unusual spin-state transitions in the reduction of ferrylmyoglobin at low temperature

Ferrylmyoglobin is reduced in γ-irradiated aqueous ethylene glycol solutions at 77 K to form a novel ferric low-spin type compound. The transition of the low-temperature product to the final high-spin ferrimyoglobin at higher temperature involves several intermediate species appearing in a sequence as temperature increases. With manifestation of the transient low-spin forms and probably the mid-spin S = 3 2 state, the process of ferrylmyoglobin reduction is found to display the spin-state transitions scarcely observed in ferric heme reactions.