Hydrogen peroxide-metmyoglobin reaction

The reaction of peroxidase (metmyoglobin) with hydrogen peroxide leads to the generation of a green-blue radical from a colorless compound 2,2 -azino-(u s(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). It is slowed down in the presence of an antioxidant, an effect that is used for its quantitation in the Total Antioxidant Status Kit (manufacturer Randox, UK) [10]. Problems associated with this method are due to potential interference of the reaction compound H202 with components of the sample to be investigated. No investigation of fat-soluble compounds is possible. 

Other proteins such as methemoglobin and metmyoglobin have been observed to produce molecular oxygen in the presence of hydrogen peroxide, but at a very low rate (5). This may simply be a property of the heme, which can promote a low-level catalatic reaction in the absence of protein. Consequently, it is possible that all heme-containing proteins may exhibit catalatic reactions if assayed carefully, but such minor, largely nonquantifiable activities are not considered here. 

The reaction of other minor or type D catalases such as methemoglo-bin and metmyoglobin is not treated in detail here, because they are minor activities, significantly lower than even that of chloroperoxidase. The orientation of residues on the distal side of the heme is not optimized for the catalatic reaction to the extent that there is even a sixth ligand of the heme, a histidine, that would preclude a close association of the heme with hydrogen peroxide without a significant side-chain movement. It is only after an extended treatment with H2O2 and oxidation of the Fe that a low level of catalatic activity becomes evident. 

Fig. 4.5. ESR spectrum of the ferryl myoglobin radical (Turner et al., 1990). ESR spectrum observed on reaction of (a) metmyoglobin (225 //M) and (b) oxymyoglobin with 250 / M hydrogen peroxide in the presence of 25 mM DMPO at pH 7.4 under normoxic...

George, P. and D. H. Irvine. 1952. The reaction between metmyoglobin and hydrogen peroxide. Biochemical Journal 52 511-517. 

Whether lipid oxidation in muscle foods is catalysed by the iron redox cycle or by formation of the ferryl ions is not clear. However, ferrous ions react with lipid hydroperoxides much faster than with hydrogen peroxide. As shown above, if the reaction of metmyoglobin with hydroperoxides produces ferryl radicals capable of initiating lipid oxidation, it is necessary to prevent the formation of metmyoglobin or methemoglobin. At acidic pH, ferric myoglobin can initiate lipid oxidation in the presence of lipid hydroperoxides. 

To date, a number of procedures have been proposed for the production of ABTS + from ABTS in solutions by using chemical reactions with a variety of oxidants. The original assay developed by Miller and Rice-Evans (sometimes referred to as TEAC I) utilized the metmyoglobin—hydrogen peroxide system to produce a highly reactive intermediate (HO ), to which ABTS donates an electron, generating ABTS + [12,22]. In this case. 

Compound III, the red compound found by Keilin and Mann (1937) to be produced in the presence of excess hydrogen peroxide, was not implicated in this reaction mechanism. Although these workers state that compound III is also reduced by added donors, according to Theorell (1947) it reacts more slowly than compound II. Alkyl peroxides cannot replace hydrogen peroxide in this reaction, and there is an analogy with metmyoglobin, which reacts as showm in Eq. (5) (Keilin and Hartree, 1954). 

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