Adelaide Faljoni-Alario

Generation of active oxygen species during coupled autoxidation of oxyhemoglobin and δ-aminolevulinic acid

The conversion of oxyhemoglobin to methemoglobin has been shown via spectrophotometric, circular dichroism and polarographic studies to be accelerated by delta-aminolevulinic acid, a major heme-precursor accumulated in a number of heme-linked pathologies. Concomitantly, delta-aminolevulinic acid undergoes aerobic oxidation. The intermediacy of oxygen radicals in these processes was evidenced by the inhibitory effect of catalase, superoxide dismutase and mannitol. These results are relevant to the exacerbated production of active oxygen species in intermittent acute porphyria and saturnism carriers.

Oxidative damage to ferritin by 5-aminolevulinic acid.

5-Aminolevulinic acid (ALA), a heme precursor overproduced in various porphyric disorders, has been implicated in iron-mediated oxidative damage to biomolecules and cell structures. From previous observations of ferritin iron release by ALA, we investigated the ability of ALA to cause oxidative damage to ferritin apoprotein. Incubation of horse spleen ferritin (HoSF) with ALA caused alterations in the ferritin circular dichroism spectrum (loss of a alpha-helix content) and altered electrophoretic behavior. Incubation of human liver, spleen, and heart ferritins with ALA substantially decreased antibody recognition (51, 60, and 28% for liver, spleen, and heart, respectively). Incubation of apoferritin with 1-10mM ALA produced dose-dependent decreases in tryptophan fluorescence (11-35% after 5h), and a partial depletion of protein thiols (18% after 24h) despite substantial removal of catalytic iron. The loss of tryptophan fluorescence was inhibited 35% by 50mM mannitol, suggesting participation of hydroxyl radicals. The damage to apoferritin had no effect on ferroxidase activity, but produced a 61% decrease in iron uptake ability. The results suggest a local autocatalytic interaction among ALA, ferritin, and oxygen, catalyzed by endogenous iron and phosphate, that causes site-specific damage to the ferritin protein and impaired iron sequestration. These data together with previous findings that ALA overload causes iron mobilization in brain and liver of rats may help explain organ-specific toxicities and carcinogenicity of ALA in experimental animals and patients with porphyria.

Modulation of cytochrome c spin states by lipid acyl chains: a continuous- wave electron paramagnetic resonance (CW-EPR) study of haem iron

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.

Antioxidant and pro-oxidant properties of some di-Schiff base copper(II) complexes

Abstract The di-Schiff base complexes, [ N , N ′-bis(2-pyridyl)methylene-1,2-diaminoethane]- ( N , N ′, N ″, N ′′′)-copper(II) ( 1 ), and [ N , N ′-bis(2-acetylpyrazyl)methylene-1,3-diaminopropane]-( N , N ′, N ″, N ′′′)-copper(II) ( 2 ), were prepared in the perchlorate form, and characterized using elemental analysis, UV/Vis, infrared (IR) and electronic paramagnetic resonance (EPR) spectroscopies. The relative thermodynamic stabilities of these complexes were estimated by circular dichroism (CD), using bovine albumin as the competitive ligand. Their antioxidant properties were verified by their catalytic activities in the dismutation of superoxide radicals (SOD activity), and in the disproportionation of hydrogen peroxide (catalase activity). The determined SOD-activities were comparable to that of known SOD mimics, with IC 50 =0.447 for ( 1 ) and 0.0902 μM for ( 2 ), in comparison to 0.0054 μM for the native Cu 2 Zn 2 -SOD. The determined rate constants for the catalase activity were k =0.098 mol −1 dm 3 s −1 , and 1.10 mol −1 dm 3 s −1 to compounds ( 1 ) and ( 2 ), respectively.

Spectroscopic, structural, and functional characterization of the alternative low-spin state of horse heart cytochrome C.

The alternative low-spin states of Fe(3+) and Fe(2+) cytochrome c induced by SDS or AOT/hexane reverse micelles exhibited the heme group in a less rhombic symmetry and were characterized by electron paramagnetic resonance, UV-visible, CD, magnetic CD, fluorescence, and Raman resonance. Consistent with the replacement of Met(80) by another strong field ligand at the sixth heme iron coordination position, Fe(3+) ALSScytc exhibited 1-nm Soret band blue shift and epsilon enhancement accompanied by disappearance of the 695-nm charge transfer band. The Raman resonance, CD, and magnetic CD spectra of Fe(3+) and Fe(2+) ALSScytc exhibited significant changes suggestive of alterations in the heme iron microenvironment and conformation and should not be assigned to unfold because the Trp(59) fluorescence remained quenched by the neighboring heme group. ALSScytc was obtained with His(33) and His(26) carboxyethoxylated horse cytochrome c and with tuna cytochrome c (His(33) replaced by Asn) pointing out Lys(79) as the probable heme iron ligand. Fe(3+) ALSScytc retained the capacity to cleave tert-butylhydroperoxide and to be reduced by dithiothreitol and diphenylacetaldehyde but not by ascorbate. Compatible with a more open heme crevice, ALSScytc exhibited a redox potential approximately 200 mV lower than the wild-type protein (+220 mV) and was more susceptible to the attack of free radicals.

ELECTRONICALLY EXCITED SPECIES IN THE PEROXIDASE CATALYZED OXIDATION OF INDOLEACETIC ACID. EFFECT UPON DNA AND RNA

Abstract— The electronically excited species generated in the peroxidase (oxidase) catalyzed oxidation of the plant hormone indole‐3‐acetic acid is an excited state of indole‐3‐carboxaldehyde.

Biological Activities of Violacein, a New Antitumoral Indole Derivative, in an Inclusion Complex with β-Cyclodextrin

Violacein is a poorly water-soluble antitumoraland antibacterial drug. The solubility can be enhanced by complexation withβ-cyclodextrin. The inclusion complex was prepared by the co-precipitation method in molarratios of 1 : 1 and 1 : 2 of violacein/β-cyclodextrin, respectively. The acutetoxicity (E. coli strain) of violacein did not changeup to 400 μM, either in thepresence or absence of cyclodextrin. Cytotoxicity (V-79 cellculture) through DNA and MTT assays was significantlydecreased in the presence of the 1 : 2 molar ratio complex.Studies on erythrocyte lipid peroxidation by the thiobarbituricacid (TBA) methodshowed that violacein and violacein/β-CD (1 : 2) at100 μM cause 50% and 80% inhibition, respectively. At 500 μM theviolacein/β-CD complexinhibited lipid peroxidation completely; however, withfree violacein only 65% inhibition was reached at that concentration.

Modifications in heme iron of free and vesicle bound cytochrome c by tert-butyl hydroperoxide: a magnetic circular dichroism and electron paramagnetic resonance investigation

To characterize changes to the heme and the influence of membrane lipids in the reaction of cytochrome c with peroxides, we studied the reaction of cytochrome c with tert-butyl hydroperoxide (tert-BuOOH) by magnetic circular dichroism (MCD) and direct electron paramagnetic resonance (EPR) in the presence and absence of different liposomes. Direct low-temperature (11 degrees K) EPR analysis of the cytochrome c heme iron on exposure to tert-BuOOH shows a gradual (180 s) conversion of the low-spin form to a high-spin Fe(III) species of rhombic symmetry (g = 4.3), with disappearance of a prior peroxyl radical signal (g(o) = 2.014). The conversion to high spin precedes Soret band bleaching, observable by UV/Vis spectroscopy and by magnetic circular dichroism (MCD) at room temperature, that indicates loss of iron coordination by the porphyrin ring. The presence of cardiolipin-containing liposomes delayed formation of the peroxyl radical and conversion to high-spin iron, while dicetylphosphate (DCP) liposomes accelerated these changes. Correspondingly, bleaching of cytochrome c by tert-BuOOH at room temperature was accelerated by several negatively charged liposome preparations, and inhibited by mitochondrial-mimetic phosphatidylcholinephosphatidylethanolaminecardiolipin (PCPECL) liposomes. Concomitant with bleaching, spin-trapping measurements with 5,5-dimethyl-1-pyroline-N-oxide showed that while the relative production of peroxyl, alkoxyl, and alkyl radicals was unaffected by DCP liposomes, PCPECL liposomes decreased the spin-trapped alkoxyl radical signal by 50%. The EPR results show that the primary initial change on exposure of cytochrome c to tert-BuOOH is a change to a high-spin Fe(III) species, and together with MCD measurements show that unsaturated cardiolipin-containing lipid membranes influence the interaction of tert-BuOOH with cytochrome c heme iron, to alter radical production and decrease damage to the cytochrome.

LIPOSOME EFFECT ON THE CYTOCHROME C-CATALYZED PEROXIDATION OF CARBONYL SUBSTRATES TO TRIPLET SPECIES

Cytochrome c exhibits peroxidase activity on diphenylacetaldehyde (DPAA) and 3-methylacetoacetone (MAA), which is greatly affected by the presence and nature of charged liposome or micelle interfaces interacting with the enzyme. The ferricytochrome c reaction with DPAA is accelerated when the enzyme is attached to negatively charged interfaces. Whatever the medium, bulk solution or negatively charged dicetylphosphate (DCP), phosphatidylcholine/phosphatidylethanolamine/cardiolipin (PC/PE/CL) liposomes, this chemiluminescent reaction is accompanied by reduction of cytochrome c to its ferrous form. In turn, MAA is oxidized by cytochrome c exclusively when bound to DCP liposomes. Contrary to DPAA oxidation, the MAA reaction is followed by bleaching of cytochrome c, reflecting damage to the hemeprotein chromophore. The cytochrome-c-catalyzed oxidation of either DPAA or MAA leads to concomitant disappearance of the enzyme charge transfer absorption band at 695 nm. This suggests that the peroxidase activity of cytochrome c involves substrate-induced loss of the methionine ligand at the iron sixth coordination position, which is favored by interaction of cytochrome c with negatively charged interfaces. Accordingly, a decrease and blue shift of the charge transfer band could be observed in cytochrome-c-containing negatively charged DCP, PC/PE/CL liposomes or lysophosphatidylethanolamine micelles in the presence of DPAA or MAA.

A CIRCULAR DICHROISM AND FLUORESCENCE QUENCHING STUDY OF THE INTERACTIONS BETWEEN RHODIUM(II) COMPLEXES AND HUMAN SERUM ALBUMIN

Various divalent rhodium complexes Rh2(L)4 (L = acetate, propionate, butyrate, trifluoroacetate and trifluoroacetamidate) have been found to bind to non-defatted human serum albumin (HSA) at molar ratios about 8:1. The circular dichroism measurements showed that the more liposoluble carboxylates, butyrate and trifluoroacetate, caused the major alterations of the secondary structure of HSA. Stern-Volmer constants for the fluorescence quenching of the buried Trp214 residue by these complexes were also higher for the lipophilic metal compounds. In the case of the rhodium carboxylates it was observed that their denaturating and quenching properties could be explained in terms of their liposolubilities: the higher their lipophilic characters, the higher their abilities to penetrate inside the protein framework leading to structural alterations, and the closer they could get to the Trp residue causing fluorescence quenching. The liposoluble amidate complex, Rh2 (tfc)4, presented an intermediate quenching and did not cause structural alterations in the protein, presumably not penetrating inside the peptidic backbone. This study shows that it is possible to design new antitumor metal complexes which bind, to a large extent, to a transport protein causing little structural damage.