The Peroxidase Activities Of Protein-heme Complexes And Their Catalytic Activities For Protein Nitration And Oxidation

Heme (Fe-protoporphyrin Ⅸ) is one of the protein cofactors frequently used in nature to achieve such diverse functions. In some pathological states, heme is released from hemoglobin and probably binds with other protein or biomolecules. The different ways of proteins binding with heme make different effects on its peroxidase activities. Free heme promotes lipid peroxidation, and has been suggested to contribute to protein and DNA damage. Tyrosine nitration catalyzed by heme containing proteins in the presence of nitrite and H2O2is one of the important pathways for protein nitration in vivo. Protein nitration is a marker of oxidative stress occurring in the pathogenesis of numerous diseases, including neurodegenerative diseases, T2Dm and cardiovascular pathologies. In addition, protein carbonylation, the most common irreversible reaction of protein oxidation, occurs inevitably in the oxidative damage in the heme-H2O2-NO2-system. Therefore, the impact of protein binding with heme on its peroxidase activity is related to the toxic effect of the complex on coexistent protein. In this paper, we studied the peroxidase activities of heme proteins and its effects on the coexistent protein:(1) The binding properties of insulin and heme and the effects of which on the peroxidase activity of heme. Here, we investigated the complexation of heme with insulin and the peroxidase activity of the complexes. The data showed that insulin could complex with heme and increase the pseudo-peroxidase activity of heme. Heme-insulin complex catalyzed H2O2and NO2-to nitrate tyrosine more effectively than free heme, which may be related to the elevated protein nitration in beta cells. Besides, the tyrosine residues of insulin were also oxidized to form tyrosine radical cross-linkage in the presence of H2O2, leading to covalent aggregation of insulin which would affect the hormone’s bioactivity. This study indicated that heme-insulin may be a previously unrecognized contributor to the dysfunction of beta cells and the pathogenesis of T2Dm.(2) The peroxidase activity of HSA-heme and its function. The data showed that the peroxidase activity of heme increased upon HSA binding. HSA-heme showed higher efficiency in catalyzing tyrosine oxidation than free heme in the presence of H2O2. Methemalbumin promoted self-nitration and nitration of tyrosine in coexistent protein, but decreased carbonylation of coexistent protein compared with heme. The heme to protein cross-linked forms of methemalbumin suggested that HSA trapped the free radical accompanied by the formation of ferryl heme. When tyrosine residues in HSA were modified by iodination, HSA lost of protection effect on protein carbonylation. The low concentration of glutathione could effectively inhibit tyrosine nitration, but had no effect on protein carbonylation. Taken together, these observations indicate that HSA enhances heme peroxidase activity but that HSA transfers the free radical to tyrosine residues of HSA, forming tyrosyl radicals to protect surrounding proteins from oxidation, and the increased tyrosine radicals can be reduced by endogenic antioxidant such as GSH.(3) The study on the interaction of GAPDH and heme and the function of GAPDH-heme. The effect on the coexistent protein and the peroxidase activity of GAPDH-heme complex were compared with HSA-heme complex. The result showed that binding with GAPDH could inhibit H2O2-mediated degradation of heme. The peroxidase activity of GAPDH-heme was higher than free heme, but significantly lower than HSA-heme. However, the order of catalytic activity of heme and its complex toward tyrosine oxidation/nitration was, HSA-heme> heme> GAPDH-heme. It indicated that GAPDH-heme complex is selective toward substrates and it could reduce the nitrative stress in the presence of H2O2and NO2-. The carbonylation of coexistent protein was also decreased by GAPDH-heme, but the carbonylation of GAPDH was increased by incubation with heme-H2O2-NO2-system. The protection of GAPDH to heme-GO-NO2- induced cytotoxicity in SH-SY5Y cells was more effective than that of HSA. In addition, the formation of heme to protein cross-linked species was not detected in GAPDH-heme complexes after reaction with H2O2, which may be result from the little protein radical formation in GAPDH. These observations indicate that binding with GAPDH inhibit the activity of heme in catalyzing tyrosine nitration and protect the coexistent protein from oxidative damage, and the mechanism was different from HSA. This study may help clarify the protective role of GAPDH acting as a chaperone in heme transfer to downstream areas.