Comparative reactivities of various biological compounds with myeloperoxidase-hydrogen peroxide-chloride,and similarity of oxidant to hypochlorite

The reactivities of myeloperoxidase-H₂O₂-Cl^? and sodium hypochlorite with amino acids, uric acid, NADH, ascorbic acid, ADP, albumin, haemoglobin, α₁-antitrypsin and some hydroxyl radical scavengers have been compared. The ability of each compound to inhibit chlorination of monochlorodimedon by both oxidants was measured. Relative reaction rates varied over a range of 10⁵, but the reactivities of the two oxidants with each compound were very similar, from which it is concluded that the reactions of hypochlorite accurately reflect those of the myeloperoxidase system. Thiol compounds (cysteine and GSH) and methionine were more than 100-times more reactive than other amino acids, which had comparable reactivity to NADH and uric acid. Benzoate, dimethylsulphoxide and formate were very much less reactive. The significance of these reactions of myeloperoxidase in microbial killing and inflammation is discussed.     

Oxidized amino acids: culprits in human atherosclerosis and indicators of oxidative stress

Oxidized low-density lipoprotein (LDL) is implicated in atherogenesis, but the mechanisms that oxidize LDL in the human artery wall have proven difficult to identify. A powerful investigative approach is mass spectrometric quantification of the oxidized amino acids that are left in proteins by specific oxidation reactions. Comparison of these molecular fingerprints in biological samples with those produced in proteins by various in vitro oxidation systems can indicate which biochemical pathway has created damage in vivo. For example, the pattern of oxidized amino acids in proteins isolated from atherosclerotic lesions implicates reactive intermediates generated by myeloperoxidase, a major phagocyte enzyme. These intermediates include hypochlorous acid, tyrosyl radical, and reactive nitrogen species, each of which generates a different pattern of stable end products. Despite this strong evidence that myeloperoxidase promotes LDL oxidation in vivo, the antioxidant that has been tested most extensively in clinical trials, vitamin E, fails to inhibit myeloperoxidase pathways in vitro. Because the utility of an antioxidant depends critically on the nature of the pathway that inflicts tissue damage, interventions that specifically inhibit myeloperoxidase or other physiologically relevant pathways would be more logical candidates for the prevention of cardiovascular disease. Moreover, levels of oxidized amino acids in urine and plasma might reflect those in tissues and therefore identify individuals with high levels of oxidative stress. Trials with such subjects would seem more likely to uncover effective antioxidant therapies than trials involving the general population.     

The reaction of 2,4,6-trinitrobenzenesulphonic acid with amino acids, peptides and proteins

1. The kinetics of the reaction of 2,4,6-trinitrobenzenesulphonic acid with various amino acids, peptides and proteins were studied by spectrophotometry. 2. The reaction of the α- and -amino groups in simple amino acids was found to be second-order, and the unprotonated amino group was shown to be the reactive species. 3. By allowing for the concentration of unreactive −NH₃⁺ group, intrinsic reactivities for the free amino groups were derived and shown to be correlated with the basicities. 4. The SH group of N-acetylcysteine was found to be more reactive to 2,4,6-trinitrobenzenesulphonic acid than most amino groups. 5. The reactions of insulin, chymotrypsinogen and ribonuclease with 2,4,6-trinitrobenzenesulphonic acid were analysed in terms of three exponential rate curves, each referring to one or more amino groups of the proteins. 6. The reaction of lysozyme with 2,4,6-trinitrobenzenesulphonic acid was found to display an acceleration effect. 7. From the reaction of 2,4,6-trinitrobenzenesulphonic acid with glutamate dehydrogenase at several enzyme concentrations, it was possible to discern two sets of amino groups of different reactivity, and to show that the number of groups in each set was decreased by aggregation of the enzyme.     

Chlorohydrin formation from unsaturated fatty acids reacted with hypochlorous acid.

Stimulated neutrophils produce hypochlorous acid (HOCl) via the myeloperoxidase-catalyzed reaction of hydrogen peroxide with chloride. The reactions of HOCl with oleic, linoleic, and arachidonic acids both as free fatty acids or bound in phosphatidylcholine have been studied. The products were identified by gas chromatography-mass spectrometry of the methylated and trimethylsilylated derivatives. Oleic acid was converted to the two 9,10-chlorohydrin isomers in near stoichiometric yield. Linoleic acid, at low HOCl:fatty acid ratios, yielded predominantly a mixture of the four possible monochlorohydrin isomers. Bischlorohydrins were also formed, in increasing amounts at higher HOCl concentrations. Arachidonic acid gave a complex mixture of mono- and bischlorohydrins, the relative proportions depending on the amount of HOCl added. Linoleic acid appears to be slightly more reactive than oleic acid with HOCl. Reactions of oleic and linoleic acids with myeloperoxidase, hydrogen peroxide, and chloride gave chlorohydrin products identical to those with HOCl. Lipid chlorohydrins have received little attention as products of reactions of neutrophil oxidants. They are more polar than the parent fatty acids, and if formed in cell membranes could cause disruption to membrane structure. Since cellular targets for HOCl appear to be membrane constituents, chlorohydrin formation from unsaturated lipids could be significant in neutrophil-mediated cytotoxicity.     

Initiation of a superoxide-dependent chain oxidation of lactate dehydrogenase-bound NADH by oxidants of low and high reactivity

In cells, NADH and NADPH are mainly bound to dehydrogenases such as lactate dehydrogenase (LDH). In cell-free systems, the binary LDH-NADH complex has been demonstrated to produce reactive oxygen species via a chain oxidation of NADH initiated and propagated by superoxide. We studied here whether this chain radical reaction can be initiated by oxidants other than LDH largely increased the oxidation of NADH (but not of NADPH) by O(2), H(2)O(2) and during the intermediacy of HNO(2). LDH also increased the oxidation of NADH by peroxynitrite. The increases in NADH oxidation were completely prevented by superoxide dismutase (SOD). In contrast, the nitrogen dioxide-dependent oxidation of NADH and NADPH was decreased by LDH in a SOD-independent manner. These experimental data strongly indicate that oxidation of LDH-bound NADH can be induced from reaction of either weak oxidants with LDH-bound NADH or of strong oxidants with free NADH thus yielding which is highly effective to propagate the chain. Our results underline the importance of SOD in terminating superoxide-dependent chain reactions in cells under oxidative stress.     

Reactions of Peroxynitrite with Uric Acid: Formation of Reactive Intermediates,Alkylated Products and Triuret,and In Vivo Production of Triuret Under Conditions of Oxidative Stress

Hyperuricemia is associated with hypertension, metabolic syndrome, preeclampsia, cardio-vascular disease and renal disease, all conditions associated with oxidative stress. We hypothesized that uric acid, a known antioxidant, might become prooxidative following its reaction with oxidants; and, thereby contribute to the pathogenesis of these diseases. Uric acid and 1,3-¹⁵N₂-uric acid were reacted with peroxynitrite in different buffers and in the presence of alcohols, antioxidants and in human plasma. The reaction products were identified using liquid chromatography-mass spectrometry (LC-MS) analyses. The reactions generate reactive intermediates that yielded triuret as their final product. We also found that the antioxidant, ascorbate, could partially prevent this reaction. Whereas triuret was preferentially generated by the reactions in aqueous buffers, when uric acid or 1,3-¹⁵N₂-uric acid was reacted with peroxynitrite in the presence of alcohols, it yielded alkylated alcohols as the final product. By extension, this reaction can alkylate other biomolecules containing OH groups and others containing labile hydrogens. Triuret was also found to be elevated in the urine of subjects with preeclampsia, a pregnancy-specific hypertensive syndrome that is associated with oxidative stress, whereas very little triuret is produced in normal healthy volunteers. We conclude that under conditions of oxidative stress, uric acid can form reactive intermediates, including potential alkylating species, by reacting with peroxynitrite. These reactive intermediates could possibly explain how uric acid contributes to the pathogenesis of diseases such as the metabolic syndrome and hypertension.     

The effect of thyroxine and related compounds on the aerobic myeloperoxidase--catalysed oxidation of NADH

Thyroxine concentrations as low as 1 microM significantly stimulate compound III formation during aerobic oxidation of NADH by highly purified myeloperoxidase. This increased compound III formation is paralleled by an increased oxidation of NADH. Stimulation of various thyronine and tyrosine analogues was in the order T4 greater than T3 greater than 3,5-T2 (or triiodothyropropionic acid). Thyronine and diiodotyrosine had no significant effect. From the potencies of the various thyronines to stimulate compound III formation, the following structural features seem necessary: (1) Substitution of thyronine with four iodine atoms. (2) An amino group on the alanine side chain. (3) Both aromatic rings of thyronine.     

Urate causes the human polymorphonuclear leukocyte to secrete superoxide.

The human polymorphonuclear leukocyte generates O2-. and H2O2 when it is treated with uric acid. A transition metal catalyzed reaction between O2-. and H2O2 can give the hydroxyl radical and myeloperoxidase forms hypochlorous acid from H2O2 and chloride. Therefore, the uric acid-induced secretion of oxidants may be responsible for a large part of the inflammation associated with gout.     

Kinetics of the reactions of hypochlorous acid and amino acid chloramines with thiols, methionine, and ascorbate

Thiol oxidation by hypochlorous acid and chloramines is a favorable reaction and may be responsible for alterations in regulatory or signaling pathways in cells exposed to neutrophil oxidants. In order to establish the mechanism for such changes, it is necessary to appreciate whether these oxidants are selective for different thiols as compared with other scavengers. We have measured rate constants for reactions of amino acid chloramines with a range of thiols, methionine, and ascorbate, using a combination of stopped-flow and competitive kinetics. For HOCl, rate constants are too fast to measure directly by our system and values relative to reduced glutathione were determined by competition with methionine. For taurine chloramine, the rate constants for reaction with 5-thio-2-nitrobenzoic acid, GSH, methionine, and ascorbate at pH 7.4 were 970, 115, 39, and 13 M(-1) s(-1), respectively. Values for 10 thiols varied by a factor of 20 and showed an inverse relationship to the pK(a) of the thiol group. Rate constants for chloramines of glycine and N-alpha-acetyl-lysine also showed these relationships. Rates increased with decreasing pH, suggesting a mechanism involving acid catalysis. For hypochlorous acid, rates of reaction with 5-thio-2-nitrobenzoic acid, GSH, cysteine, and most of the other thiols were very similar. Relative reactivities varied by less than 5 and there was no dependence on thiol pK(a). Chloramines have the potential to be selective for different cellular thiols depending on their pK(a). For HOCl to be selective, other factors must be important, or its reactions could be secondary to chloramine formation.     

Reaction of β-propiolactone with amino acids and its specificity for methionine

1. The reactions of beta-propiolactone with amino acids were investigated under various conditions of pH and temperature to find those under which the reagent acted with specificity. 2. At pH9.0 and 22 degrees , after 15min. of reaction, at least 85% of each amino acid had reacted, methionine and cystine being the most reactive. 3. At pH7.0 and 22 degrees most amino acids reacted; methionine, cystine and histidine reacted almost entirely, and proline and lysine to a significantly smaller extent. 4. At pH3.0 and 22 degrees further specificity was obtained; methionine and cystine were the only reactive amino acids. 5. Reaction at pH3.0 and 0 degrees was specific for methionine; it was the only amino acid modified even after 145hr. of reaction.     

Interrelation between thrombin structure and its stability

Temperature inactivation of human thrombin has been studied when finding out the mechanism of this enzyme stabilization by amino acids. Effect of a number of amino acids on thrombin in the conditions (pH) of the highest activity of proteinase has been investigated. It is established that most amino acids are characterized to more or less extent by the protective action, when hampering the temperature inactivation of the enzyme. The correspondence was mainly found between the stabilizing effect of amino acids and thrombin specificity. Thrombin is stabilized by L-arginine and DL-lysine more intensively than by other amino acids. A stabilizing effect of L-glutamic acid was shown in contrast to the action of the latter on trypsin that was obviously connected with the original structure of the active centre of thrombin, that is the availability of anionic binding centre which includes Lys68, Arg72, Arg77. High thrombin stabilization by such amino acids as phenylalanine, DL-serine, DL-methonine was an exception. It was established that amino acids stabilize thrombin with formation of a compound with the reactive centre of its molecule, like the compounds enzyme-substrate. The macrostructure stability probably depends, to a considerable extent, on the state of the enzyme reactive centre: thrombin molecules, which contain a free reactive centre, are more labile than those which reactive centre is bound to the reagent of more or less specific character. The inhibition of the autolysis process may be another manifestation of thrombin stabilization by amino acids.     

Effect of reducing-equivalent disposal and NADH/NAD on deamination of amino acids by intact rumen microorganisms and their cell extracts.

When mixed rumen microorganisms were incubated in media containing the amino acid source Trypticase, both monensin and carbon monoxide (a hydrogenase inhibitor) decreased methane formation and amino acid fermentation. Both of the methane inhibitors caused a significant increase in the ratio of intracellular NADH to NAD. Studies with cell extracts of rumen bacteria and protozoa indicated that the ratio of NADH to NAD had a marked effect on the deamination of reduced amino acids, in particular branched-chain amino acids. Deamination was inhibited by the addition of NADH and was stimulated by methylene blue, an agent that oxidizes NADH. Neutral and oxidized amino acids were unaffected by NADH. The addition of small amounts of 2-oxoglutarate greatly enhanced the deamination of branched-chain amino acids and indicated that transamination via glutamate dehydrogenase was important. Formation of ammonia from glutamate was likewise inhibited by NADH. These experiments indicated that reducing-equivalent disposal and intracellular NADH/NAD ratio were important effectors of branched-chain amino acid fermentation.     

Effect of reducing-equivalent disposal and NADH/NAD on deamination of amino acids by intact rumen microorganisms and their cell extracts

When mixed rumen microorganisms were incubated in media containing the amino acid source Trypticase, both monensin and carbon monoxide (a hydrogenase inhibitor) decreased methane formation and amino acid fermentation. Both of the methane inhibitors caused a significant increase in the ratio of intracellular NADH to NAD. Studies with cell extracts of rumen bacteria and protozoa indicated that the ratio of NADH to NAD had a marked effect on the deamination of reduced amino acids, in particular branched-chain amino acids. Deamination was inhibited by the addition of NADH and was stimulated by methylene blue, an agent that oxidizes NADH. Neutral and oxidized amino acids were unaffected by NADH. The addition of small amounts of 2-oxoglutarate greatly enhanced the deamination of branched-chain amino acids and indicated that transamination via glutamate dehydrogenase was important. Formation of ammonia from glutamate was likewise inhibited by NADH. These experiments indicated that reducing-equivalent disposal and intracellular NADH/NAD ratio were important effectors of branched-chain amino acid fermentation.     

Chemical basis of inflammation-induced carcinogenesis

Chronic inflammation induced by biological, chemical, and physical factors has been associated with increased risk of human cancer at various sites. Inflammation activates a variety of inflammatory cells, which induce and activate several oxidant-generating enzymes such as NADPH oxidase, inducible nitric oxide synthase, myeloperoxidase, and eosinophil peroxidase. These enzymes produce high concentrations of diverse free radicals and oxidants including superoxide anion, nitric oxide, nitroxyl, nitrogen dioxide, hydrogen peroxide, hypochlorous acid, and hypobromous acid, which react with each other to generate other more potent reactive oxygen and nitrogen species such as peroxynitrite. These species can damage DNA, RNA, lipids, and proteins by nitration, oxidation, chlorination, and bromination reactions, leading to increased mutations and altered functions of enzymes and proteins (e.g., activation of oncogene products and/or inhibition of tumor-suppressor proteins) and thus contributing to the multistage carcinogenesis process. Appropriate treatment of inflammation should be explored further for chemoprevention of human cancers.     

Enhanced oxidation of NAD(P)H by oxidants in the presence of dehydrogenases but no evidence for a superoxide-propagated chain oxidation of the bound coenzymes

Recently we demonstrated that lactate dehydrogenase (LDH)-bound NADH is oxidized by O2, H2O2, HNO2 and peroxynitrite predominantly via a chain radical mechanism which is propagated by superoxide. Here we studied both whether other dehydrogenases also increase their coenzymes' reactivity towards these oxidants and whether a chain radical mechanism is operating. Almost all dehydrogenases increased the oxidation of their physiological coenzymes by at least one of the oxidants. The oxidation of NADH or NADPH depended both on the binding dehydrogenase and the applied oxidant and in some cases the reactions were remarkably fast. The highest rate constant (k = 370 M-1 s-1) was found for the reaction of HNO2 with NADH bound to alcohol dehydrogenase. Regardless of the applied oxidant, superoxide dismutase failed to inhibit the oxidation of protein-bound NADH and NADPH. We therefore conclude that several dehydrogenases increase the oxidation of NADH and/or NADPH by the employed set of oxidants in bimolecular reactions, but, unlike LDH, do not mediate a O2*(-) dependent chain radical mechanism.     

Comparative transport activity of intact cells, membrane vesicles, and mesosomes of Bacillus licheniformis

Sodium ion was shown to stimulate strongly the transport of l-glutamic acid into cells of Bacillus licheniformis 6346 His(-). Lithium ion had a slight capacity to replace Na(+) in this capacity, but K(+) was without effect. Three of five amino acids tested. l-glutamic acid, l-aspartic acid, and l-alanine, were concentrated against a gradient in the cells. Intracellular pools of these amino acids were extractable with 5% trichloroacetic acid. Pools of l-histidine and l-lysine could not be detected. No evidence of active transport of lysine into cells could be detected, and histidine was taken up in the absence of chloramphenicol but not in its presence. The uptake of glutamic acid by membrane vesicle preparations was strongly stimulated by reduced nicotinamide adenine dinucleotide (NADH) and to a lesser extent by succinate. The presence of phenazine methosulfate increased uptake in the presence of succinate. Either l- or d-lactate and adenosine triphosphate were without effect. None of these compounds stimulated the uptake of glutamic acid by mesosomes, although some mesosome preparations contained separable membrane which was very active. NADH strongly stimulated the uptake of aspartic acid and alanine by membrane vesicles but had only a slight effect on the uptake of histidine and lysine. No evidence of active transport of any of the amino acids into mesosomes could be detected either in the presence or absence of NADH. NADH stimulation of the uptake of glutamic acid by membrane vesicles was destroyed by exposure to light of 360 nm; this inactivation was reversible by vitamin K(2(5)) or K(2(10)). Sodium ion stimulated transport of glutamic acid by membrane vesicles.     

Hypochlorous acid as a precursor of free radicals in living systems

Hypochlorous acid (HOCl) is produced in the human body by the family of mammalian heme peroxidases, mainly by myeloperoxidase, which is secreted by neutrophils and monocytes at sites of inflammation. This review discusses the reactions that occur between HOCl and the major classes of biologically important molecules (amino acids, proteins, nucleotides, nucleic acids, carbohydrates, lipids, and inorganic substances) to form free radicals. The generation of such free radical intermediates by HOCl and other reactive halogen species is accompanied by the development of halogenative stress, which causes a number of socially important diseases, such as cardiovascular, neurodegenerative, infectious, and other diseases usually associated with inflammatory response and characterized by the appearance of biomarkers of myeloperoxidase and halogenative stress. Investigations aimed at elucidating the mechanisms regulating the activity of enzyme systems that are responsible for the production of reactive halogen species are a crucial step in opening possibilities for control of the development of the body’s inflammatory response.     

Comparative effect of N-substituted dehydroamino acids and α-tocopherol on rat liver lipid peroxidation activities

Free radical damage has been associated with a growing number of diseases and conditions, such as autoimmune diseases, neurodegenerative disorders and multiple types of cancer. Some dehydroamino acids and corresponding peptides can function as radical scavengers. In this study the in vitro effects on rat liver lipid peroxidation levels of fourteen N-substituted dehydroamino acid derivatives and α-tocopherol were investigated. α-Tocopherol is a powerful antioxidant that is beneficial in the treatment of many free radical related diseases. The results indicated that all the compounds showed very good inhibitory effect on the lipid peroxidation compound with α-tocopherol at 1 mM concentrations and the inhibition rate was in the range of 70–79 % with the exception of compound 5. At 0.1 mM concentrations compounds 1, 2 and 9 were found more active than α-tocopherol. The results confirmed that molecules such as dehydroamino acids which have reactive double bonds can act as a guard in vitro against oxidants.     

Action of biologically-relevant oxidizing species upon uric acid. Identification of uric acid oxidation products

Uric acid is an end-product of purine metabolism in Man, and has been suggested to act as an antioxidant in vivo. Products of attack upon uric acid by various oxidants were measured by high performance liquid chromatography. Hypochlorous acid rapidly oxidized uric acid, forming allantoin, oxonic/oxaluric and parabanic acids, as well as several unidentified products. HOCl could oxidize all these products further. Hydrogen peroxide did not oxidize uric acid at detectable rates, although it rapidly oxidized oxonic acid and slowly oxidized allantoin and parabanic acids. Hydroxyl radicals generated by hypoxanthine/xanthine oxidase or Fe2(+)-EDTA/H2O2 systems also oxidized uric acid to allantoin, oxonic/oxaluric acid and traces of parabanic acid. Addition of ascorbic acid to the Fe2(+)-EDTA/H2O2 system did not increase formation of oxidation products from uric acid, possibly because ascorbic acid can 'repair' the radicals resulting from initial attack of hydroxyl radicals upon uric acid. Mixtures of methaemoglobin or metmyoglobin and H2O2 also oxidized uric acid: allantoin was the major product, but some parabanic and oxonic/oxaluric acids were also produced. Caeruloplasmin did not oxidize uric acid under physiological conditions, although simple copper (Cu2+) ions could, but this was prevented by albumin or histidine. The possibility of using oxidation products of uric acid, such as allantoin, as an index of oxidant generation in vivo in humans is discussed.     

Transient kinetics of reduction of blue copper proteins by free flavin and flavodoxin semiquinones

Rate constants have been determined for the electron-transfer reactions between reduced free flavins and flavodoxin semiquinone and several blue copper proteins. Correlations between these values and redox potentials demonstrate that spinach plastocyanin, Pseudomonas aeruginosa azurin, Alcaligenes sp. azurin, and Alcaligenes sp. nitrite reductase have the same intrinsic reactivities toward free flavins, whereas stellacyanin is more reactive (3.3 times) and laccase considerably less reactive (approximately 12 times). Electrostatic interactions between the negatively charged flavin mononucleotide (FMN) and the copper proteins show that the interaction site charges for laccase and nitrite reductase are opposite in sign to the net protein charge and that the signs and magnitudes of the charges are consistent with the known three-dimensional structures for plastocyanin and the azurins and with amino acid sequence homologies for stellacyanin. The results demonstrate that the apparent interaction site charge with flavodoxin is larger than that with FMN for plastocyanin, nitrite reductase, and stellacyanin but smaller for Pseudomonas azurin. This is interpreted in terms of a larger interaction domain for the flavodoxin reaction, which allows charged groups more distant from the actual electron-transfer site to become involved. The intrinsic reactivities of plastocyanin and azurin toward flavodoxin are the same, as was the case with FMN, but both stellacyanin and nitrite reductase are considerably less reactive than expected (approximately 2 orders of magnitude). This result suggests the involvement of steric factors with these latter two proteins which discriminate against large reactants such as flavodoxin.