Hemoproteins denaturation

Denaturation of hemoproteins in cooked meats leads to liberation of the heme and oxidation of the porphyrin ring. Nonheme iron is less available nutritionally than heme iron and affects lipid oxidation more. In methemoglo-bin and metmyoglobin solutions heated for one hour at 78°C and 100°C the degradation of heme was about 22 to 26%, while after two hours at 120°C it increased to about 85 to 95% (Oellingrath, 1988). In meat cookery, however, such severe conditions do not apply. 

Since hemoproteins such as lactoperoxidase and catalase are inhibited more rapidly than the sulfhydryl oxidation occurs, it is unlikely that the rapid activation of guanylate cyclase occurs by sulfhydryl oxidation [132]. Prolonged incubation of the papain or dehydrogenase enzymes with substrate and nitroprusside yielded a turbidity which indicated denaturation of the enzyme to an insoluble form, possibly by the formation of disulfide bridges via the dimerization of thiyl radicals [132]. 

Fig. 3. Axial ligands of the heme iron in hemoproteins. The imidazole ring of a histidyl residue is one of the axial ligands in myoglobin, hemoglobin, and cytochrome c. In native cytochrome c the sixth ligand is a methionyl residue of the polypeptide chain. In partially denatured cytochrome c, and in myoglobin and hemoglobin, a variety of ligands, some of which are shown in the figure, may bind to the sixth coordination site...

Alves, O.C. Wajnberg, E. Low temperature photolysis of denatured nitrosyl hemoproteins. Int. J. Biol. Macromol. 1998, 23, 157-164. 

Cytochrome P450 monooxygenases, which are protoheme proteins, present an unusually red-shifted Soret band for the reduced CO complex at 450 nm [167]. Upon denaturation by various treatments, the cytochromes are always able to bind CO reversibly by losing their unique spectral properties as well as their catalytic activities. The spectral properties of the reduced carbonylated complexes of the denaturated form called P420 are identical to those of the dioxygen-carrying hemoproteins (Amax = 420 nm). These facts indicate that the unusual spectral properties of cytochrome P450 closely relate to its function. 

Early ORD measurements on beef liver catalase showed large Cotton effects in the Soret region, which disappeared upon denaturation (255). No evidence for previously reported (256) Cotton effects near 600 nm could be obtained (255). ORD and CD measurements on bovine liver catalase included also the liganded hemoprotein. Conformational changes in the protein, occurring upon ligand addition, were deduced from data in the ultraviolet region (257). 

In studying the reaction of the heme group in hemoproteins it is always necessary to ensure that no alteration in protein structure is affecting the results. Complete denaturation is easily recognized by precipitation and flocculation and the liberation of heme of hematin, but minor changes can also occur. In the case of peroxidase two forms were isolated by Theorell (25) and named peroxidase I and II. The first of these, now called paraperoxidase (Theorell, 26), has been found to have undergone some alteration possibly the removal of the carbohydrate portion of the protein molecule or possibly reversible denaturation to a slight extent (Keilin and Hartree, 48). The paraperoxidase shows enzymatic activity comparable to that of the intact enzyme. 

As discussed before in the case of nucleic acids the authors have also considered the incidence of the interfacial conformation of the hemoproteins on the appearance of the SERRS signals from the chromophores. Although under their Raman conditions no protein vibration can be observed, the possibility of heme loss or protein denatura-tion are envisaged to explain a direct interaction of the heme chromophores with the electrode surface in the case of the adsorl Mb. extensive denaturation of Cytc at the electrode appears unlikely to the authors on the basis of the close correspondence of the surface and solution spectra. Furthermore, the sluggish electron transfer kinetics measured by cyclic voltammetry in the case of Cytc is also an argument in favour of some structural hindrance for the accessibility to the heme chromophore in the adsorbed state of Cytc. This electrochemical aspect of the behaviour of Cytc has very recently incited Cotton et al. and Tanigushi et al. to modify the silver and gold electrode surface in order to accelerate the electron transfer. The authors show that in the presence of 4,4-bipyridine bis (4-pyridyl)disulfide and purine an enhancement of the quasi-reversible redox process is possible. The SERRS spectroscopy has also permitted the characterization of the surface of the modified silver electrode. It has teen thus shown, that in presence of both pyridine derivates the direct adsorption of the heme chromophore is not detected while in presence of purine a coadsorption of Cytc and purine occurs In the case of the Ag-bipyridyl modified electrode the cyclicvoltammetric and SERRS data indicate that the bipyridyl forms an Ag(I) complex on Ag electrodes with the appropriate redox potential to mediate electron transfer between the electrode and cytochrome c. 

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