[show abstract][hide abstract] ABSTRACT: The aim of this work was to study the effects of P. major against the oxidative damage of isolated rat liver mitochondria.
The extracts were obtained using methanol (MeOH), ethyl acetate (EAc), dichloromethane (DCM), and hexane (Hex) as solvents.
Hex, DCM, and EAc totally, and MeOH partially, inhibited ROS generation and lipid peroxidation of membranes induced by Fe(2+) or t-BOOH. However, only MeOH was able to prevent the t-BOOH-induced glutathione and NAD(P)H oxidation. All extracts chelated Fe(2+) and reduced DPP Hradicals. EPR analysis revealed that P. major exhibited potent scavenger activity for hydroxyl radicals.
The potent antioxidant activity exhibited by P. major was able to prevent oxidative mitochondrial damage, contributing to the understanding of its hepatoprotective action against ROS-mediated toxicity.
The Journal of pharmacy and pharmacology. 08/2012; 64(8):1177-87.
[show abstract][hide abstract] ABSTRACT: Aerobic organisms are afforded with an antioxidant enzymatic apparatus that more recently has been recognized to include cytochrome c, as it is able to prevent hydrogen peroxide generation by returning electrons from the superoxide ion back to the respiratory chain. The present study investigated the glutathione peroxidase (GPx), superoxide dismutase (SOD) and cytochrome c-like antioxidant activities of para Mn(III)TMPyP in isolated rat liver mitochondria (RLM) and mitoplasts. In RLM, Mn(III)TMPyP decreased the lipid-peroxide content associated with glutathione (GSH) depletion consistent with the use of GSH as a reducing agent for high valence states of Mn(III)TMPyP. SOD and cytochrome c antioxidant activities were also investigated. Mn(II)TMPyP was able to reduce ferric cytochrome c, indicating the potential to remove a superoxide ion by returning electrons back to the respiratory chain. In antimicyn A-poisoned mitoplasts, Mn(III)TMPyP efficiently decreased the EPR signal of DMPO-OH adduct concomitant with GSH depletion. The present results are consistent with SOD and GPx activities for Mn(III)TMPyP and do not exclude cytochrome c-like activity. However, considering that para Mn(III)TMPyP more efficiently reduces, rather than oxidizes, superoxide ion; electron transfer from the Mn(II)TMPyP to the respiratory chain might not significantly contribute to the superoxide ion removal, since most of Mn(II)TMPyP is expected to be produced at the expense of NADPH/GSH oxidation. The present results suggest GPx-like activity to be the principal antioxidant mechanism of Mn(III)TMPyP, whose efficiency is dependent on the NADPH/GSH content in cells.
Journal of Bioenergetics 12/2011; 43(6):663-71. · 1.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: AbstractThe effects of nitrosative species on cyt c structure and peroxidase activity were investigated here in the presence of O2•- and anionic and zwitterionic vesicles. Nitrosative species were generated by 3-morpholinesydnonymine (SIN1) decomposition, using cyt c heme iron and/or molecular oxygen as electron acceptor. Far- and near-UV CD spectra of SIN1-treated cyt c revealed respectively a slight decrease of α-helix content (from 39 to 34%) and changes in the tryptophan structure accompanied by increased fluorescence. The Soret CD spectra displayed a significant decrease of the positive signal at 403 nm. EPR spectra revealed the presence of a low-spin cyt c form (S = 1/2) with g1 = 2.736, g2 = 2.465, and g3 = 2.058 after incubation with SIN1. These data suggest that the concomitant presence of NO• and O2•- generated from dissolved oxygen, in a system containing cyt c and liposomes, promotes chemical and conformational modifications in cyt c, resulting in a hypothetical bis-histidine hexacoordinated heme iron. We also show that, paradoxically, O2•- prevents not only membrane lipoperoxidation by peroxide-derived radicals but also oxidation of cyt c itself due to the ability of O2•- to reduce heme iron. Finally, lipoperoxidation measurements showed that, although it is a more efficient peroxidase, SIN1-treated cyt c is not more effective than native cyt c in promoting damage to anionic liposomes in the presence of tert-ButylOOH, probably due to loss of affinity with negatively charged lipids.
[show abstract][hide abstract] ABSTRACT: The encapsulation of microperoxidases (MPs) into molecular sieves with controlled pore size, such as the mesoporous silica MCM-41, represents a nanotechnology strategy to control the catalytic properties of MPs and mimic the enzymatic activity of hemoproteins. In this work, the ferric microperoxidase-11 (MP-11), obtained from trypsin-catalyzed hydrolysis of horse-heart cytochrome c, was entrapped in MCM-41, thus resulting in a catalyst (Fe(III)MP11MCM41) with catalase and monooxygenase properties. The entrapment of MP-11 inside MCM-41 was confirmed by elemental analysis and UV-visible spectrum, with a red shift in the Soret band indicating that the heme group was in a hydrophobic microenvironment. Similarly to catalase, the catalyst Fe(III)MP11MCM41 exhibited specificity for hydrogen peroxide to be converted to a high-valence oxidized intermediate, Compound II. Also mimicking catalase, the cleavage of hydrogen peroxide by MP11MCM41 resulted in O2 production detected by a Clark electrode. Phenol was able to act as reducing agent of MP11MCM41 Compound II leading to the completion of a peroxidase cycle, as confirmed by UV-visible spectrometry and EPR measurements. The analysis of the reaction products by high performance liquid chromatogram coupled to tandem mass spectrometry (HPLC/MS) revealed 2,4-dihydroxyphenol as the product of phenol oxidation by MP11MCM41. Therefore, in addition to catalase activity, the catalyst MP11MCM41 also displayed monooxygenase properties, which was possible because the MP-11 heme iron promotes homolytic cleavage of the hydrogen peroxide generating hydroxyl radicals. With such characteristics, MCM-41-entrapped MP-11 is a promising catalyst for nanobiotechnological devices.
Journal of Nanoscience and Nanotechnology 11/2007; 7(10):3643-52. · 1.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: Microperoxidases (MP) as water-soluble models attract interest to studying the reaction mechanism of peroxidases because these heme peptides are able to form the same enzyme intermediates during the reaction with peroxides. In this work we have demonstrated that the association of Fe(III)MP-9 and Fe(III)MP-11 with CTAB micelles (MP-9/CTAB and MP11/CTAB) provides a microenvironment with an alkaline interface and a hydrophobic core that exhibits peroxidase behavior. This microenvironment shifts positively the redox potential of microperoxidases by approximately 100 mV. tert-Butylhydroperoxide (t-BuOOH) when added to the medium, converted Fe(III)MP-9/CTAB to MP-9/CTAB Compound II, a high valence oxidized intermediate of the heme peptide. Subsequent addition of diphenylacetaldehyde (DPAA) to MP-9/CTAB Compound II regenerated the native form of the enzyme, Fe(III)MP-9/CTAB, what characterizes the occurrence of a peroxidase cycle. Fe(III)MP-9/CTAB regenerated during the peroxidase cycle reacted with residual DPAA in the medium to form Fe(II)MP-9/CTAB, which indicates that both Fe(III)MP-9/CTAB and its oxyferryl form can use aldehydes as reducing agents. According to the determined reduction potential, Fe(III)MP-9 and Fe(III)MP-9/CTAB should be able to oxidize DPAA (reduction potential -630 mV). The reaction of MP-9/CTAB with DPAA produced benzophenone as final product, detected by infrared spectroscopy and mass spectrometry. Interestingly, a significant difference was observed in the benzophenone yield according to the micelle/MP-9 molar ratio.
Physical Chemistry Chemical Physics 05/2006; 8(16):1963-73. · 3.83 Impact Factor
[show abstract][hide abstract] ABSTRACT: The low spin states of microperoxidases (MP)-8, -9 and -9 N-acetylated (N-Ac) were characterized using UV-visible, circular dichroism, and electron paramagnetic resonance spectroscopies over the 6.0-12.0 pH range. The first MP-8 alkaline transition (pK(a)=8.53) produced hemepeptide aggregates in the low spin state in which a water molecule was replaced by the peptide chain N-terminal group of a neighboring MP-8 molecule. Higher pH led to the deprotonation of the MP-8 histidine imidazole ring (pK(a)=10.37) at the fifth coordination position. This MP-8 species was in equilibrium with a high spin state aggregate in which OH(-) replaced histidinate, the histidinate becoming the heme iron sixth ligand in a neighboring MP-8 molecule. In a similar way to the N-AcMP-8, the low spin state of N-AcMP-9 was produced by the deprotonation of the water molecule (pK(a)=9.6) situated at the sixth coordination position of the heme iron. Up to pH 8.5, the low spin states of MP-9 were aggregates in which the alpha-amino group of Lys13 replaced water at the sixth coordination position of a neighboring MP-9 molecule. Above pH 8.5, the epsilon-amino groups of Lys13 established intra-chain coordination and impaired the formation of aggregates. Such intra-chain interaction in MP9 was supported by molecular dynamics simulation. These MP-9 monomers might also exhibit OH(-) or histidinate at the fifth coordination position.
Journal of Inorganic Biochemistry 03/2006; 100(2):226-38. · 3.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: Microperoxidases are attractive water-soluble models to study the reaction mechanism of peroxidases because they are heme
peptides able to form the same enzyme intermediates along the reaction with peroxides. In this work, we have demonstrated
that cetyltrimethylammonium bromide micelles provide a microenvironment with an alkaline interface and a hydrophobic core
that favors the peroxidase activity of microperoxidase-9. The addition of tert-butyl hydroperoxide to a system containing microperoxidase-9/cetyltrimethylammonium bromide micelles led to the occurrence
of a peroxidase cycle, indicating that this aggregate behaves as a true enzyme. This lipoenzyme is an interesting model for
studies of the peroxidase reaction mechanism and for applications in nanotechnology.
KeywordsMicroperoxidase-9–Horseradish peroxidase cycle–
t-Butyl hydroperoxide–Free radicals
[show abstract][hide abstract] ABSTRACT: Literature data have pointed to microperoxidase-8 (MP-8) as an attractive water-soluble model for studying the reaction mechanism of peroxidases because this heme peptide is able to form the same enzyme intermediates during reaction with peroxides. In this work, we have demonstrated that the association of Fe(III)MP-8 with CTAB micelles provides a microenvironment with an alkaline interface and a hydrophobic core that gives special characteristics to the Fe(III)MP-8/peroxide (tert-butyl hydroperoxide or hydrogen peroxide) reaction as compared with homogeneous medium. EPR spin-trapping studies using 5,5-dimethyl-1-pyrroline N-oxide and computer simulations of the experimental spectra were performed to determine the reaction mechanism. From the analysis of the results, alkoxyl and hydroxyl radicals of t-BuOOH and HOOH, respectively, were identified as the initial radicals produced, presumably by homolytic scission of the O−O bond by Fe(III)MP-8/CTAB. The UV−vis spectral changes for Fe(III)MP-8 pointed to the formation of Compound II as the species that exhibits subsequent bleaching. The peculiarity of the CTAB micellar microenvironment allowed circumvention of rapid kinetics, permitting the reaction to be accompanied on the scale of seconds. The Km and the maximal conversion rate (k2) of CTAB-bound Fe(III)MP-8 into the corresponding Compound II were determined for the reaction with peroxides in 20 mM CTAB, at pH 7.4 and 9.1. For both substrates, the Km values increased at pH 9.1 without significant changes in k2 values, indicating alteration in the affinity of the substrates for CTAB-bound Fe(III)MP-8.
The Journal of Physical Chemistry B 06/2004; 108(30):11124–11132. · 3.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: This work is a systematic study, showing a clear correlation between the nature of the lipid acyl chain and the spin states of cytochrome c interacting with different types of lipid membranes. According to the lipid acyl chain type, and the head group charge present in the bilayer, three spin states of cytochrome c were observed in different proportions: the native cytochrome c low spin state with rhombic symmetry (spin 1/2, g axially=3.07 and g radially=2.23), a low spin state with less rhombic symmetry (spin 1/2, g(1)=2.902, g(2)=2.225, and g(3)=1.510) and the high spin state (spin 5/2, g axially=6.0 and g radially=2.0). The proportion of the spin states of cytochrome c bound to bilayers was also dependent on the lipid/protein ratio, suggesting the existence of two or more protein sites interacting with the lipids. The lipid-induced alterations in the symmetry and spin states of cytochrome c exhibited partial reversibility when the ionic strength was increased, which reinforces the crucial role played by the electrostatic interaction with the lipid bilayer. Different cytochrome c spin states exhibited corresponding modifications in the haemprotein UV/visible spectra, particularly in the Q-band associated with loss of the 695 nm band and appearance of a band in the region of 600-650 nm. The observed reactivity of cytochrome c with oxidized forms of unsaturated lipids reinforces the possibility of the acyl chain insertion in the haemprotein structure.