Protein Radical Formation Research Articles

Effects of irradiation on natural antioxidants of cinnamon ( Cinnamomum zeylanicum N.)

Food irradiation to reduce the number of spoilage microorganisms and insects is an ionizing process that induces free radical formation in proteins, lipids, carbohydrates and other molecular structures in food. Antioxidants generally decrease the level of oxidation in such systems by transferring hydrogen atoms to the free radical structure. In the present paper, the effect of ionizing radiation on natural cinnamon antioxidants is studied. Cinnamon samples were purchased from retailers and irradiated with a 60Co source, Gammacell 220 (A.E.C.L.) installed at IPEN (São Paulo, Brazil) using 0, 5, 10, 15, 20, 25 kGy at room temperature. After irradiation 3 kinds of sequential extractions were performed. One was submitted to antioxidant extraction using ethyl ether, the second with ethanol and the last with water. The antioxidant activity was determined by β-carotene/linoleic acid co-oxidation. Irradiation in the dose range applied did not have any effect on the antioxidant potential of the cinnamon compounds. Further studies will be performed to study the possibility to use cinnamon extracts in preserving food from oxidative damage induced by ionizing radiation.

Protein Radical Formation during Lactoperoxidase-mediated Oxidation of the Suicide Substrate Glutathione: IMMUNOCHEMICAL DETECTION OF A LACTOPEROXIDASE RADICAL-DERIVED 5,5-DIMETHYL-1-PYRROLINE N-OXIDE NITRONE ADDUCT

A novel anti-5,5-dimethyl-1-pyrroline N-oxide (DMPO) polyclonal antiserum that specifically recognizes protein radical-derived DMPO nitrone adducts has been developed. In this study, we employed this new approach, which combines the specificity of spin trapping and the sensitivity of antigen-antibody interactions, to investigate protein radical formation from lactoperoxidase (LPO). When LPO reacted with GSH in the presence of DMPO, we detected an LPO radical-derived DMPO nitrone adduct using enzyme-linked immunosorbent assay and Western blotting. The formation of this nitrone adduct depended on the concentrations of GSH, LPO, and DMPO as well as pH values, and GSH could not be replaced by H(2)O(2). The level of this nitrone adduct was decreased significantly by azide, catalase, ascorbate, iodide, thiocyanate, phenol, or nitrite. However, its formation was unaffected by chemical modification of free cysteine, tyrosine, and tryptophan residues on LPO. ESR spectra showed that a glutathiyl radical was formed from the LPO/GSH/DMPO system, but no protein radical adduct could be detected by ESR. Its formation was decreased by azide, catalase, ascorbate, iodide, or thiocyanate, whereas phenol or nitrite increased it. GSH caused marked changes in the spectrum of compound II of LPO, indicating that GSH binds to the heme of compound II, whereas phenol or nitrite prevented these changes and reduced compound II back to the native enzyme. GSH also dose-dependently inhibited the peroxidase activity of LPO as determined by measuring 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) oxidation. Taken together, these results demonstrate that the GSH-dependent LPO radical formation is mediated by the glutathiyl radical, possibly via the reaction of the glutathiyl radical with the heme of compound II to form a heme-centered radical trapped by DMPO.

UVA-ketoprofen-induced hemoglobin radicals detected by immuno-spin trapping.

Ketoprofen (3-benzoyl-alpha-methylbenzeneacetic acid, KP) is a widely used nonsteroidal anti-inflammatory drug (NSAID) that causes both phototoxicity and photoallergy. Here, we investigated the formation of hemoglobin radicals, in both purified hemoglobin and red blood cells (RBC), induced by ultraviolet A (UVA)-KP by using "immuno-spin trapping," a novel approach that combines the specificity of spin trapping with the sensitivity of antigen-antibody interactions. The methemoglobin (metHb) radicals react covalently with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) to form nitroxyl radical adducts that are oxidized to the corresponding nitrone adducts, which in turn are specifically recognized by antiserum against DMPO nitrone. We found that the formation of nitrone adducts in metHb depended on the UVA dose, the KP concentration and the presence of DMPO, as determined by enzyme-linked immunosorbent assay and Western blotting. Adduct formation decreased when irradiation was carried out in the presence of catalase or nitrogen, suggesting that H2O2 plays a key role in KP-UVA-induced metHb radical formation. KP in the dark did not generate metHb radical-derived nitrone adducts, whereas UVA alone resulted in the formation of metHb radical-derived nitrone adducts that increased with UVA dose from 4 to 10 J/cm2. However, KP (25 and 200 microM) plus UVA (4 and 10 J/cm2) resulted in a significant increase in the formation of metHb radical-derived nitrone adducts as compared with UVA or KP alone, indicating that KP photosensitized the production of the metHb radicals in the presence of UVA. In contrast, no metHb radical-derived nitrone adduct was detected in the absence of DMPO, even though KP and UVA were present. We also detected the hemoglobin radical formation in RBC as well as in hemolysates. The endogenous antioxidants and exogenous reduced glutathione inhibited the protein radical formation. These studies have shown that the immuno-spin-trapping technique can be used to detect radical damage in proteins as a result of photosensitizing reactions. The successful detection of protein radical formation caused by KP photosensitization could help further understand the photoallergic effect of this NSAID.

Activated neutrophils oxidize extracellular proteins of endothelial cells in culture: effect of nitric oxide donors.

Protein oxidation of human umbilical-vein endothelial cells (HUVEC) in culture was examined under various conditions of oxidative stress. Extracellular protein (ECP) oxidation was assessed by determining dityrosine bond formation, which is indicated by the covalent coupling of the membrane-impermeable tyramine-fluorescein conjugate (TyrFluo) to HUVEC proteins. The acetylated membrane-permeable form of TyrFluo (acetylTyrFluo) was used for the determination of intracellular protein (ICP) oxidation. Oxidative stress was induced by exposing the HUVEC to PMA-activated human neutrophils, to a horseradish peroxidase/hydrogen peroxide (HRP/H(2)O(2)) system or to H(2)O(2) alone. Coupling of the probes was determined by confocal laser scanning microscopy and by Western blotting using anti-fluorescein antibody. Diethylamine nitric oxide (DEANO) was used to determine the effect of NO on the tyrosyl radical formation in proteins. The oxidative burst generated by activated neutrophils for 15 min, resulted in inducing dityrosine formation in ECP of HUVEC. Similar results were obtained with HRP/H(2)O(2), but H(2)O(2) alone did not have any effect on ECP. In the presence of DEANO (0.1 mM or higher), ECP oxidation was almost completely inhibited. This indicates that NO may protect endothelial cells against protein oxidation by activated neutrophils under pro-inflammatory conditions. Activated neutrophils did not oxidize ICP of HUVEC, which strongly suggests that the effect of the oxidative burst was restricted to the proteins exposed to the medium.

Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods.

A novel protein superfamily with over 600 members was discovered by iterative profile searches and analyzed with powerful bioinformatics and information visualization methods. Evidence exists that these proteins generate a radical species by reductive cleavage of S:-adenosylmethionine (SAM) through an unusual Fe-S center. The superfamily (named here Radical SAM) provides evidence that radical-based catalysis is important in a number of previously well- studied but unresolved biochemical pathways and reflects an ancient conserved mechanistic approach to difficult chemistries. Radical SAM proteins catalyze diverse reactions, including unusual methylations, isomerization, sulfur insertion, ring formation, anaerobic oxidation and protein radical formation. They function in DNA precursor, vitamin, cofactor, antibiotic and herbicide biosynthesis and in biodegradation pathways. One eukaryotic member is interferon-inducible and is considered a candidate drug target for osteoporosis; another is observed to bind the neuronal Cdk5 activator protein. Five defining members not previously recognized as homologs are lysine 2,3-aminomutase, biotin synthase, lipoic acid synthase and the activating enzymes for pyruvate formate-lyase and anaerobic ribonucleotide reductase. Two functional predictions for unknown proteins are made based on integrating other data types such as motif, domain, operon and biochemical pathway into an organized view of similarity relationships.

Lactoperoxidase-induced protein oxidation in milk.

The reaction between lactoperoxidase (LPO) and H(2)O(2) in the presence of bovine serum albumin (BSA), beta-lactoglobulin, or casein was investigated for the formation of protein radicals by freeze-quench electron spin resonance (ESR) and by the formation of the protein oxidation product, dityrosine. The presence of BSA resulted in a dramatic change after 1 min of reaction in the obtained ESR spectrum compared with the spectrum obtained for LPO and H(2)O(2) alone. Furthermore, experiments employing BSA or beta-lactoglobulin resulted in the formation of long-lived protein radicals detectable 10 min after initiation of the reaction. The presence of casein resulted in a minor change in the fine structure of the ESR spectrum after 1 min of reaction compared with LPO and H(2)O(2) alone, but no difference between the two reaction mixtures could be observed after 10 min of reaction. The formation of dityrosine could be detected in reaction mixtures containing LPO and H(2)O(2) after 1 and 10 min of incubation at 25 degrees C both in the absence and in the presence of BSA, beta-lactoglobulin, or casein. The presence of casein resulted in an increased dityrosine concentration compared with the reaction with LPO and H(2)O(2) alone. Endogenous LPO in unpasteurized milk was activated at 25 degrees C by adding 1 mM H(2)O(2). Radical species could be detected directly in the milk by freeze-quench ESR during the initial phase of the reaction, and dityrosine could be measured after 4 h of incubation. The role of LPO activity in the formation of ESR detectable radical species and dityrosine in milk was further verified in ultrahigh temperature (UHT) milk with no endogenous enzyme activity, as the formation of ESR detectable radical species and dityrosine took place in UHT milk only upon the addition of both H(2)O(2) and exogenous LPO.

One-electron oxidation pathway of peroxynitrite decomposition in human blood plasma: evidence for the formation of protein tryptophan-centred radicals.

Exposure of human blood plasma to peroxynitrite in the presence of 3,5-dibromo-4-nitrosobenzenesulphonic acid (DBNBS) resulted in the trapping of a strongly immobilized nitroxide radical adduct. The adduct was due to protein-centred radicals derived not only from serum albumin but also from other major plasma proteins (fibrinogen, IgG, alpha1-antitrypsin and transferrin). Urate significantly protected plasma from the peroxynitrite-induced DBNBS-plasma protein adduct, whereas ascorbate and glutathione were protective at concentrations exceeding those usually found in plasma. Alkylation of plasma -SH groups did not affect the intensity of DBNBS-plasma protein adduct, whereas bicarbonate increased its formation, thus showing a pro-oxidant effect. The DBNBS-plasma protein adduct provided little structural information, but subsequent non-specific-protease treatment resulted in the detection of an isotropic three-line spectrum, indicating the trapping of radicals centred on a tertiary carbon. The nitrogen hyperfine coupling constant of this adduct and its superhyperfine structure were similar to those of DBNBS-tryptophan peptides with the alpha-amino group of tryptophan linked in the amide bond, consistent with a radical adduct formed at C-3 of the indole ring of tryptophan-containing peptides. DBNBS was unable to trap radicals derived from peroxynitrite-treated tyrosine or tyrosine-containing peptides. Methionine treated with peroxynitrite resulted in the trapping of at least two DBNBS-methionine adducts with hyperfine structures different from that of protease-treated DBNBS-plasma proteins. These results demonstrate that peroxynitrite induced in blood plasma the formation of protein radicals centred on tryptophan residues and underline the relevance of the one-electron oxidation pathway of peroxynitrite decomposition in biological fluids.

Formation of Long-Lived Protein Radicals in the Reaction Between H 2O 2-Activated Metmyoglobin and Other Proteins

Free radicals formed during the reaction of H 2O 2 and metmyoglobin in the presence of bovine serum albumin (BSA) were investigated using freeze quench and spin-trap ESR spectroscopy. Increasing concentrations of BSA (0–300 μM) resulted in drastic changes in the characteristic freeze quench ESR signal of H 2O 2-activated metmyoglobin (perferryl protein radical) under physiological conditions (pH = 7.4; I = 0.16). The radical species formed during reaction of 100 μM H 2O 2, 100 μM metmyoglobin, and 200 μM BSA have half-lives of approximately 13 min at 25°C, in contrast to the perferryl protein radical that has a half-life of approximately 28 s at 25°C. The radical species formed in the presence of BSA were reactive towards ascorbate, glutathione, cysteine, and tyrosine. Substitution of BSA with defatted BSA, γ-globulin or β-lactoglobulin also resulted in formation of long-lived free radical species (half-lives: 13–18 min); however, the ability to form these was dependent of the specific protein and decreased in the following order: BSA > defatted BSA > γ-globulin > β-lactoglobulin. The spin-trap α-phenyl- tert-butylnitrone (PBN) showed the presence of transient protein radical species formed in the reaction between MMb, H 2O 2, and BSA. Transient radical species that could be proposed as intermediates in the formation of the long-lived protein radicals detected by freeze-quench ESR. Dityrosine was formed in the reaction between MMb, H 2O 2, and BSA, showing the involvement of tyrosine residues in the present reaction. The described chemical interaction between H 2O 2-activated myoglobin and other proteins have major consequences on future interpretations of the significance of the perferryl protein radical in biological systems where proteins are abundant.

Conformational changes induced in bovine serum albumin by the photodynamic action of haematoporphyrin

The photodynamic action of haematoporphyrin upon bovine serum albumin, spin-labelled at the cysteine-34 residue, has been shown to: (i) increase its susceptibility to proteolysis by chymotrypsin and trypsin, and (ii) increase its susceptibility to denaturation by urea. This is thought to be the result of conformational changes caused by the formation of protein radicals, although contributions from subsequent radical reactions of, for example, amino acids, may also take place. Such species have previously been shown, by EPR spin-trapping, to be formed in this system. Increased proteolytic susceptibility of non spin-labelled protein is also observed upon photolysis with haematoporphyrin, indicating that the changes observed in the spin-labelled protein also occur in the native form, and are not artefactual in nature. The significance of these photochemically-induced conformational changes within proteins in the photodynamic therapy of tumours, and other protein-radical systems is discussed.

Heterolytic versus homolytic peroxide bond cleavage by sperm whale myoglobin and myoglobin mutants

Despite extensive work on homolytic versus heterolytic peroxide bond cleavage by iron porphyrins, the nature of the corresponding reactions catalyzed by myoglobin (Mb) and hemoglobin (Hb) remains unclear. These hemoproteins react with peroxides to give a ferryl (Fe IV =O) complex and a protein radical, but the coupling of dioxygen bond cleavage to protein radical formation is obscure. This process is examined here with the help of 4-hydroperoxy-4-methyl-2,6-di-tert-butyl-cyclohexa-2,5-dien-1-one (BHTOOH), a compound that is converted heterolytically to an alcohol and homolytically to several rearranged products

Free radical initiation in proteins and amino acids by ionizing and ultraviolet radiations and lipid oxidation--part III: free radical transfer from oxidizing lipids.

Parallels and similarities in chemical and functional damage to proteins by ionizing and uv radiations and oxidizing lipids have been recognized for some time. However, only recently have oxidizing lipids been shown directly by electron spin resonance to be radiomimetic also in their capacity for protein free radical production. Free radicals play a key role in the transformation of energy to molecular and cellular damage. It is thus of critical importance to elucidate the general mechanisms of free radical formation and reactions in proteins in order to understand protein involvement in various pathological conditions and in food deterioration. Accordingly, this review is a detailed comparison of gamma-radiation, UV radiation, and lipid oxidation for what is presently known concerning (1) the specific modes of energy deposition and free radical formation, (2) the free radicals formed in proteins and amino acids, and (3) the typical damage correlating with these radicals.

Free radical initiation in proteins and amino acids by ionizing and ultraviolet radiations and lipid oxidation--Part 22: ultraviolet radiation and photolysis.

Parallels and similarities in chemical and functional damage to proteins by ionizing and UV radiations and oxidizing lipids have been recognized for some time. However, only recently have oxidizing lipids been shown directly by electron spin resonance to be radiomimetic also in their capacity for protein free radical production. Free radicals play a key role in the transformation of energy to molecular and cellular damage. It is thus of critical importance to elucidate the general mechanisms of free radical formation and reactions in proteins in order to understand protein involvement in various pathological conditions and in food deterioration. Accordingly, this review is a detailed comparison of gamma radiation, UV radiation, and lipid oxidation for what is presently known concerning (1) the specific modes of energy deposition and free radical formation, (2) the free radicals formed in proteins and amino acids, and (3) the typical damage correlating with these radicals.

Free radical initiation in proteins and amino acids by ionizing and ultraviolet radiations and lipid oxidation--Part I: ionizing radiation.

Parallels and similarities in chemical and functional damage to proteins by ionizing and UV radiations and oxidizing lipids have been recognized for some time. However, only recently have oxidizing lipids been shown directly by electron spin resonance to be radiomimetic also in their capacity for protein free radical production. Free radicals play a key role in the transformation of energy to molecular and cellular damage. It is thus of critical importance to elucidate the general mechanisms of free radical formation and reactions in proteins in order to understand protein involvement in various pathological conditions and in food deterioration. Accordingly, this review is a detailed comparison of gamma radiation, UV radiation, and lipid oxidation for what is presently known concerning (1) the specific modes of energy deposition and free radical formation, (2) the free radicals formed in proteins and amino acids, and (3) the typical damage correlating with these radicals.

E.S.R. Study of Spin-trapped Radicals Formed during the Photolysis of Aqueous Solutions of Acid Amides and H2O2

Free radicals formed by the reactions of OH radicals with amides and their N-methylated derivatives in aqueous solutions have been studied. The OH radicals were produced by U.V.-photolysis of H2O2, and the short-lived amide radicals were converted to more stable nitroxide radicals by addition to a spin-trap, tert-nitrosobutane. The spin-trapped radicals were identified by e.s.r. spectroscopy. For acetamide, chloroacetamide, malonamide, succinamide and propionamide, the observed radicals were formed by H-abstraction from the carbon atoms attached to the carbonyl group. The H atom attached to the carbonyl group was abstracted in formamide. For N-methyl acetamide, N,N-dimethyl acetamide and the corresponding formamide derivatives, H-abstraction occurred only from the N-methyl group. The non-equivalency of the amide protons was observed in the spin-trapped radicals for acetamide, formamide, malonamide, succinamide and propionamide. The identification of the site of OH attack on N-methyl amides is helpful for the study of radical formation in peptides and proteins.

The effect of metal ions on free radical formation and reactions in irradiated proteins.

SummaryThe effect of different metal ions on free radical formation and reactions in gamma-irradiated bovine serum albumin and trypsin in the solid state has been studied using electron spin resonance spectroscopy. The metal ions lowered the radical concentration as observed at 77°k, and markedly reduced the concentration of —CH2–S· radicals formed at room temperature. The results have been interpreted in terms of the reaction of radiation-produced electrons with the metal ions, and they confirm the postulate that, at 77°k, electrons are trapped on cystine residues in proteins and that the resultant cystine anion reacts at higher temperatures to give the —CH2–S· radical. The E.S.R. results have been correlated with the results of Butler and Robins, who found that metal ions protected trypsin against high-energy radiation.

The effect of sulphur compounds on free radical reactions and formation in irradiated dry proteins.

Abstract The free radical reactions occurring in gamma-irradiated proteins: salmine, gelatin and zein, freeze-dried with mercaptoethylamine (cysteaine), cysteine or cystamine have been studied using electron spin resonance spectroscopy. It was found that at 77° K the energy was not absorbed randomly over the whole macromolecule and particularly an unpaired electron (probably an ion) was stabilised on a sulphur atom in the additive molecule. At room temperature the protein radicals migrated to the additive to give the radical, CH2S•. This radical then reacted with a glycine residue according to the reaction: − CH 2  S • + − CO  CH 2  NH − → CH 2  SH + − CO  C H  NH − so that the major effect of the added sulphur compounds was to accelerate the rate of migration of the initial protein radicals to a glycine residue. Also they accelerated the decay of total radicals in proteins.

Primary radical formation in irradiated proteins.

Primary radical formation in irradiated proteins.