On the irreversible destruction of reduced nicotinamide nucleotides by hypohalous acids

Degradation of the reduced pyridine nucleotides NMNH and NADH by HOCl involves two distinct stages: a fast reaction, k = 4.2 x 10(5) M(-1) s(-1), leads to generation of stable pyridine products (Py/Cl) with a strong absorption band at 275 nm (epsilon = 12.4 x 10(3) M(-1) cm(-1) in the case of NMNH); secondarily, a subsequent reaction of HOCl, k = 3.9 x 10(3) M(-1) s(-1), leads to a complete loss of the aromatic absorption band of the pyridine ring. HOBr and HOI(I(2)) react similarly. Apparent rate constants of the primary reactions of HOX species with NMNH at pH 7.2 increase in the order HOCl (3 x 10(5) M(-1) s(-1)) < HOBr( approximately 4 x 10(6) M(-1) s(-1)) < HOI(I(2))( approximately 6.5 x 10(7) M(-1) s(-1)). HOBr reacts fast also with the primary product Py/Br, k approximately 9 x 10(5) M(-1) s(-1), while the reactions of HOI and I(2) with Py/I are slower, approximately 1.4 x 10(3) M(-1) s(-1) and >6 x 10(3) M(-1) s(-1), respectively. Halogenation of the amide group of NMN(+) by HOX species is many orders of magnitude slower than oxidation of NMNH. Taurine inhibits HOCl-induced oxidation of NADH, but HOBr-induced oxidation is not inhibited because the taurine monobromamine rapidly oxidizes NADH, and oxidation by HOI(I(2)) is not inhibited because taurine is inert toward HOI(I(2)). Also sulfur compounds (GSH, GSSG, and methionine) are less efficient in protecting NADH against oxidation by HOBr and HOI(I(2)) than against oxidation by HOCl. The results suggest that reactions of HOBr and HOI(I(2)) in a cellular environment are much more selectively directed toward irreversible oxidation of NADH than reactions of HOCl. It is noteworthy that the rather inert N-chloramines react with iodide to generate HOI(I(2)), i.e., the most reactive and selective oxidant of reduced pyridine nucleotides. NMR investigations show that the primary stable products of the reaction between NMNH and HOCl are various isomeric chlorohydrins originating from a nonstereospecific electrophilic addition of HOCl to the C5&dbond;C6 double bond of the pyridine ring. The primary products (Py/X) of NMNH all exhibit similar absorption bands around 275 nm and are hence likely to result from analogous addition of HOX to the C5&dbond;C6 bond of the pyridine ring. Since the Py/X species are stable and inert toward endogeneous reductants like ascorbate and GSH, they may generally be useful markers for assessing the contribution of hypohalous acids to inflammatory injury.     

Oxidation of NADH by chloramines and chloramides and its activation by iodide and by tertiary amines

Irreversible oxidation of reduced nicotinamide nucleotides by neutrophil-derived halogen oxidants (HOCl, chloramines, HOBr, etc.) is likely to be a highly lethal process, because of the essential role of NAD(P)H in important cell functions such as mitochondrial electron transport, and control of the cellular thiol redox state by NADPH-dependent glutathione reductase. Chloramines (chloramine-T, NH(2)Cl, etc.) and N-chloramides (N-chlorinated cyclopeptides) react with NADH to generate the same products as HOCl, i.e., pyridine chlorohydrins, as judged from characteristic changes in the NADH absorption spectrum. Compared with the fast oxidation of NADH by HOCl, k approximately 3 x 10(5) M(-1) s(-1) at pH 7.2, the oxidation by chloramines is about five orders of magnitude slower; that by chloramides is about four orders of magnitude slower. Apparent rate constants for oxidation of NADH by chloramines increase with increasing proton or buffer concentration, consistent with general acid catalysis, but oxidation by chloramides proceeds with pH-independent kinetics. In presence of iodide the oxidation of NADH by chloramines or chloramides is faster by at least two orders of magnitude; this is due to reaction of iodide with the N-halogen to give HOI/I(2), the most reactive and selective oxidant for NADH among HOX species. Quinuclidine derivatives (QN) like 3-chloroquinuclidine and quinine are capable of catalyzing the irreversible degradation of NADH by HOCl and by chloramines; QN(+)Cl, the chain carrier of the catalytic cycle, is even more reactive toward NADH than HOCl/ClO(-) at physiological pH. Oxidation of NADH by NH(2)Br proceeds by fast, but complex, biphasic kinetics. A compilation of rate constants for interactions of reactive halogen species with various substrates is presented and the concept of selective reactivity of N-halogens is discussed.     

The vinyl ether linkages of plasmalogens are favored targets for myeloperoxidase-derived oxidants: a kinetic study

Plasmalogens, which contain a vinyl ether bond, are major phospholipids of the plasma membranes of endothelial and vascular smooth muscle cells and cardiac myocytes. These lipids, in contrast to other phospholipids, have been reported to be targets of HOCl/HOBr generated by myeloperoxidase, with elevated levels of the products of these reactions (alpha-chloro/alpha-bromo aldehydes and unsaturated lysophospholipids) having been detected in human atherosclerotic lesions. The reason(s) for the targeting of this lipid class, over other phospholipids, is poorly understood, and is examined here. It is shown that HOCl and HOBr react with a model vinyl ether (ethylene glycol vinyl ether) 200-300-fold faster ( k = 1.6 x 10 (3) and 3.5 x 10 (6) M (-1) s (-1), respectively) than with aliphatic alkenes (models of phospholipids). True plasmalogens react ca. 20-fold slower than the models. Chloramines and bromamines (from reaction of HOCl/HOBr with primary amines and alpha-amino groups) also react with vinyl ethers, unlike aliphatic alkenes, with k = 10 (-3)-10 (2) M (-1) s (-1) for chloramines (with the His side chain chloramine being the most reactive, k = 172 M (-1) s (-1)) and k = 10 (3)-10 (4) M (-1) s (-1) for bromamines. The bromamine rate constants are typically 10 (5)-10 (6) larger than those of the chloramines. Intermolecular vinyl ether oxidation by phospholipid headgroup bromamines can also occur. These kinetic data indicate that plasmalogens are significantly more susceptible to oxidation than the aliphatic alkenes of phospholipids, thereby rationalizing the detection of products from the former, but not the latter, in human atherosclerotic lesions.     

Dissociation of DNA double strand by hypohalous acids

Calf thymus DNA was treated with authentic HOCl, and hypohalous acid-generating systems. This caused a decrease in fluorescence of ethidium-DNA complexes when ethidium bromide was subsequently added to the DNA. The fluorescence continued to decrease up to 30 min after adding HOCl. Loss in fluorescence was proportional to the concentration of HOCl and was complete when a 3-fold excess of HOCl was added to the DNA. No significant decrease in the fluorescence was observed when the chlorination was carried out in the presence of a concentration of monochlorodimedone (MCD) equivalent to that of HOCl. MCD is known to react stoichiometrically with HOCl. The decrease in fluorescence was completely inhibited by H2O2, ascorbate and glutathione (GSH). We have estimated the rate constant for the reaction of HOCl with H2O, to be 1-2 x 10(5) M(-1)s(-1). When compared with authentic HOCl, HOCl-generating systems (Cl + H2O2 + MPO or chloroperoxidase) were found to be inefficient in damaging DNA. This result most likely arises because the rate constant for reaction of HOCl with H2O2 is about 1000-fold faster than that for the reaction with DNA. HOBr and HOI generating systems also had a limited ability to damage DNA. We conclude that good chlorine acceptors and antioxidants protect DNA from hypohalous acid-induced oxidative damage.     

Kinetic analysis of the reactions of hypobromous acid with protein components: implications for cellular damage and use of 3-bromotyrosine as a marker of oxidative stress

Hypohalous acids (HOX, X = Cl, Br) are produced by activated neutrophils, monocytes, eosinophils, and possibly macrophages. These oxidants react readily with biological molecules, with amino acids and proteins being major targets. Elevated levels of halogenated Tyr residues have been detected in proteins isolated from patients with atherosclerosis, asthma, and cystic fibrosis, implicating the production of HOX in these diseases. The quantitative significance of these findings requires knowledge of the kinetics of reaction of HOX with protein targets, and such data have not been previously available for HOBr. In this study, rate constants for reaction of HOBr with protein components have been determined. The second-order rate constants (22 degrees C, pH 7.4) for reaction with protein sites vary by 8 orders of magnitude and decrease in the order Cys > Trp approximately Met approximately His approximately alpha-amino > disulfide > Lys approximately Tyr > Arg > backbone amides > Gln/Asn. For most residues HOBr reacts 30-100 fold faster than HOCl, though Cys and Met residues are approximately 10-fold less reactive, and ring halogenation of Tyr is approximately 5000-fold faster. Thus, Tyr residues are more, and Cys and Met much less, important targets for HOBr than HOCl. Kinetic models have been developed to predict the targets of HOX attack on proteins and free amino acids. Overall, these results shed light on the mechanisms of cell damage induced by HOX and indicate, for example, that the 3-chloro-Tyr:3-bromo-Tyr ratio does not reflect the relative roles of HOCl and HOBr in disease processes.     

A comparative spin trapping study of hypobromous and hypochlorous acids interaction with tert-butyl hydroperoxide

We have demonstrated that hypochlorite (HOCI/OCl-) and hypobromite (HOBr/OBr-) can react with tert-butyl hydroperoxide with close rate constants (k(HOCl) = 10,8 M(-1) x s(1); k(HOBr) = 8,9 M(-1) x (s(-1)). By means of the spin trap 4-pyridyl-1-oxide-N-tert-butyl nitron we have found that both reactions proceed through decomposition of tert-butyl hydroperoxide and generation of tert-butyl peroxyl (OOC(CH3)3) and tert-butoxyl (OC(CH3)3) radicals, the ratio of their the concentrations being dependent on the concentration of tert-butyl hydroperoxide. Thus, hypobromite, similar to hypochlorite, is a precursor of free radicals produced in the reaction with organic hydroperoxides. This reaction can be of great importance in the intensification of free radical processes, namely, in lipid peroxidation at the stage of chain branching.     

The reactivity of myeloperoxidase compound I formed with hypochlorous acid

The reaction of human myeloperoxidase (MPO) with hypochlorous acid (HOCl) was investigated by conventional stopped-flow spectroscopy at pH 5, 7, and 9. In the reaction of MPO with HOCl, compound I is formed. Its formation is strongly dependent on pH. HOCl (rather than OCl-) reacts with the unprotonated enzyme in its ferric state. Apparent second-order rate constants were determined to be 8.1 x 10(7) M(-1)s(-1) (pH 5), 2.0 x 10(8) M(-1)s(-1) (pH 7) and 2.0 x 10(6) M(-1)s(-1) (pH 9) at 15 degrees C. Furthermore, the kinetics and spectra of the reactions of halides and thiocyanate and of physiologically relevant one-electron donors (ascorbate, nitrite, tyrosine and hydrogen peroxide) with this compound I were investigated using the sequential-mixing technique. The results show conclusively that the redox intermediates formed upon addition of either hydrogen peroxide or hypochlorous acid to native MPO exhibit the same spectral features and reactivities and thus are identical. In stopped-flow investigations, the MPO/HOCl system has some advantage since: (i) in contrast to H2O2, HOCl cannot function as a one-electron donor of compound I; and (ii) MPO can easily be prevented from cycling by addition of methionine as HOCl scavenger. As a consequence, the observed absorbance changes are bigger and errors in data analysis are smaller.     

Reactions of desferrioxamine with peroxynitrite-derived carbonate and nitrogen dioxide radicals

The iron chelating agent desferrioxamine inhibits peroxynitrite-mediated oxidations and attenuates nitric oxide and oxygen radical-dependent oxidative damage both in vitro and in vivo. The mechanism of protection is independent of iron chelation and has remained elusive over the past decade. Herein, stopped-flow studies revealed that desferrioxamine does not react directly with peroxynitrite. However, addition of peroxynitrite to desferrioxamine in both the absence and the presence of physiological concentrations of CO2 and under excess nitrite led to the formation of a one-electron oxidation product, the desferrioxamine nitroxide radical, consistent with desferrioxamine reacting with the peroxynitrite-derived species carbonate (CO3*-) and nitrogen dioxide (*NO2) radicals. Desferrioxamine inhibited peroxynitrite-dependent free radical-mediated processes, including tyrosine dimerization and nitration, oxyhemoglobin oxidation in the presence of CO2, and peroxynitrite plus carbonate-dependent chemiluminescence. The direct two-electron oxidation of glutathione by peroxynitrite was unaffected by desferrioxamine. The reactions of desferrioxamine with CO3*- and *NO2 were unambiguously confirmed by pulse radiolysis studies, which yielded second-order rate constants of 1.7 x 10(9) and 7.6 x 10(6) M(-1) s(-1), respectively. Desferrioxamine also reacts with tyrosyl radicals with k = 6.3 x 10(6) M(-1) s(-1). However, radical/radical combination reactions between tyrosyl radicals or of tyrosyl radical with *NO2 outcompete the reaction with desferrioxamine and computer-assisted simulations indicate that the inhibition of tyrosine oxidation can be fully explained by scavenging of the peroxynitrite-derived radicals. The results shown herein provide an alternative mechanism to account for some of the biochemical and pharmacological actions of desferrioxamine via reactions with CO3*- and *NO2 radicals.     

Mechanism of nitrite-stimulated catalysis by lactoperoxidase.

The reactions of lactoperoxidase (LPO) intermediates compound I, compound II and compound III, with nitrite (NO2(-)) were investigated. Reduction of compound I by NO2(-) was rapid (k2 = 2.3 x 10(7) M(-1) x s(-1); pH = 7.2) and compound II was not an intermediate, indicating that NO2* radicals are not produced when NO2(-) reacts with compound I. The second-order rate constant for the reaction of compound II with NO2(-) at pH = 7.2 was 3.5 x 10(5) M(-1) x s(-1). The reaction of compound III with NO2(-) exhibited saturation behaviour when the observed pseudo first-order rate constants were plotted against NO2(-) concentrations and could be quantitatively explained by the formation of a 1 : 1 ratio compound III/NO2(-) complex. The Km of compound III for NO2(-) was 1.7 x 10(-4) M and the first-order decay constant of the compound III/ NO2(-) complex was 12.5 +/- 0.6 s(-1). The second-order rate constant for the reaction of the complex with NO2(-) was 3.3 x 10(3) M(-1) x s(-1). Rate enhancement by NO2(-) does not require NO2* as a redox intermediate. NO2(-) accelerates the overall rate of catalysis by reducing compound II to the ferric state. With increasing levels of H2O2, there is an increased tendency for the catalytically dead-end intermediate compound III to form. Under these conditions, the 'rescue' reaction of NO2(-) with compound III to form compound II will maintain the peroxidatic cycle of the enzyme.     

Selenium-containing amino acids are targets for myeloperoxidase-derived hypothiocyanous acid: determination of absolute rate constants and implications for biological damage

Elevated MPO (myeloperoxidase) levels are associated with multiple human inflammatory pathologies. MPO catalyses the oxidation of Cl-, Br- and SCN- by H2O2 to generate the powerful oxidants hypochlorous acid (HOCl), hypobromous acid (HOBr) and hypothiocyanous acid (HOSCN) respectively. These species are antibacterial agents, but misplaced or excessive production is implicated in tissue damage at sites of inflammation. Unlike HOCl and HOBr, which react with multiple targets, HOSCN targets cysteine residues with considerable selectivity. In the light of this reactivity, we hypothesized that Sec (selenocysteine) residues should also be rapidly oxidized by HOSCN, as selenium atoms are better nucleophiles than sulfur. Such oxidation might inactivate critical Sec-containing cellular protective enzymes such as GPx (glutathione peroxidase) and TrxR (thioredoxin reductase). Stopped-flow kinetic studies indicate that seleno-compounds react rapidly with HOSCN with rate constants, k, in the range 2.8×10(3)-5.8×10(6) M-1·s-1 (for selenomethionine and selenocystamine respectively). These values are ~6000-fold higher than the corresponding values for H2O2, and are also considerably larger than for the reaction of HOSCN with thiols (16-fold for cysteine and 80-fold for selenocystamine). Enzyme studies indicate that GPx and TrxR, but not glutathione reductase, are inactivated by HOSCN in a concentration-dependent manner; k for GPx has been determined as ~5×105 M-1·s-1. Decomposed HOSCN did not induce inactivation. These data indicate that selenocysteine residues are oxidized rapidly by HOSCN, with this resulting in the inhibition of the critical intracellular Sec-dependent protective enzymes GPx and TrxR.     

Diffusion-limited component of reactions catalyzed by Bacillus cereus beta-lactamase I

The Bacillus cereus beta-lactamase I catalyzes the hydrolysis of a wide variety of penicillins and cephalosporins with values of k(cat)/K(m) varying over several orders of magnitude. The values of this parameter for the most reactive of these compounds, benzylpenicillin, I, and furylacryloyl-penicillin, II (k(cat)/K(m) = 2.43 x 10(7) M(-1) s(-1) and 2.35 x 10(7) M(-1) s(-1), respectively, at pH 7.0 in potassium phosphate buffer containing 0.17 M KCl, I(c) = 0.63, 25 degrees C) are decreased markedly by increasing viscosity in sucrose- or glycerol-containing buffers. The relative sensitivities to viscosity of k(cat)/K(m) values for I and for cephaloridine, III, were found to be virtually unchanged at pH 3.8 from those observed at pH 7.0. The differential effects of viscosity on the reactive vs. the sluggish [e.g., cephalothin (IV), k(cat)/K(m) = 1 x 10(4) M(-1) s(-1)] substrates support the contention that the rates of reaction of the former with the enzyme are in part diffusion controlled. Quantitative analysis gives values for the association rate constants, k(1), of 7.6 x 10(7) M(-1) s(-1), 4 x 10(7) M(-1) s(-1), and 1.1 x 10(7) M(-1) s(-1) for I, II, and III, respectively. As both reactive and sluggish substrates associate with the active site of the enzyme with relatively similar rate constants, the variation in k(cat)/K(m) values is primarily due to the variation in the partition ratios k(-1)/k(2), for the ES complex, which are 2.3, 0.77, and 30 for I, II, and III, respectively. The preceding analysis is based on direct application of the Stokes-Einstein diffusion law to enzyme kinetics. The range of applicability of this law to the diffusion of substrate size molecules and the mechanics of diffusion of ionic species through viscous solutions of sucrose vs. polymers are explored.     

*NO dissociation represents the rate limiting step for O2-mediated oxidation of ferrous nitrosylated Mycobacterium leprae truncated hemoglobin O

Mycobacterium leprae truncated hemoglobin O (trHbO) protects from nitrosative stress and sustains mycobacterial respiration. Here, kinetics of M. leprae trHbO(II)-NO denitrosylation and of O(2)-mediated oxidation of M. leprae trHbO(II)-NO are reported. Values of the first-order rate constant for *NO dissociation from M. leprae trHbO(II)-NO (k(off)) and of the first-order rate constant for O(2)-mediated oxidation of M. leprae trHbO(II)-NO (h) are 1.3 x 10(-4) s(-1) and 1.2 x 10(-4) s(-1), respectively. The coincidence of values of k(off) and h suggests that O(2)-mediated oxidation of M. leprae trHbO(II)-NO occurs with a reaction mechanism in which *NO, that is initially bound to heme(II), is displaced by O(2) but may stay trapped in a protein cavity(ies) close to heme(II). Next, M. leprae trHbO(II)-O(2) reacts with *NO giving the transient Fe(III)-OONO species preceding the formation of the final product M. leprae trHbO(III). *NO dissociation from heme(II)-NO represents the rate limiting step for O(2)-mediated oxidation of M. leprae trHbO(II)-NO.     

Kinetics and mechanism of the reduction of CrVI and CrV by D-lactobionic acid

The oxidation of D-lactobionic acid by Cr(VI) yields the 2-ketoaldobionic acid and Cr(3+) as final products when a 20-times or higher excess of the aldobionic acid over Cr(VI) is used. The redox reaction takes place through a complex multistep mechanism, which involves the formation of intermediate Cr(IV) and Cr(V) species. Cr(IV) reacts with lactobionic acid much faster than Cr(V) and Cr(VI) do, and cannot be directly detected. However, the formation of CrO(2)(2+), observed by the first time for an acid saccharide/Cr(VI) system, provides indirect evidence for the intermediacy of Cr(IV) in the reaction path. Cr(VI) and the intermediate Cr(V) react with lactobionic acid at comparable rates, being the complete rate laws for the Cr(VI) and Cr(V) consumption expressed by: -d[Cr(VI)]/dt=[k(I)+k(II)[H(+)]][lactobionicacid][Cr(VI)], where k(I)=(4.1+/-0.1) x 10(-3) M(-1) s(-1) and k(II)=(2.1+/-0.1) x 10(-2) M(-2) s(-1); and -d[Cr(V)]/dt=[k(III)[H(+)]+(k(IV)+k(V)[H(+)])[lactobionicacid]] [Cr(V)], where k(III)=(1.8+/-0.1) x 10(-3) M(-1) s(-1), k(IV)=(1.1+/-0.1) x 10(-2) M(-1) s(-1) and k(V)=(1.0+/-0.1) x 10(-2) M(-2) s(-1), at 33 degrees C. The Electron Paramagnetic Resonance (EPR) spectra show that five-co-ordinate oxo-Cr(V) bischelates are formed at pH 1-5 with the aldobionic acid bound to Cr(V) through the alpha-hydroxyacid group.     

Hypochlorite- and hypobromite-mediated radical formation and its role in cell lysis.

Activated leukocytes generate the potent oxidants HOCl and HOBr via the formation of H(2)O(2) and the release of peroxidase enzymes (myeloperoxidase, eosinophil peroxidase). HOCl and HOBr are potent microbiocidal agents, but excessive or misplaced production can cause tissue damage and cell lysis. In this study it is shown that HOBr induces red blood cell lysis at approximately 10-fold lower concentrations than HOCl, whereas with monocyte (THP1) and macrophage (J774) cells HOCl and HOBr induce lysis at similar concentrations. The role of radical formation during lysis has been investigated by EPR spin trapping, and it is shown that reaction of both oxidants with each cell type generates cell-derived radicals. Red blood cells exposed to nonlytic doses of HOCl generate novel nitrogen-centered radicals whose formation is GSH dependent. In contrast, HOBr gives rise to nitrogen-centered, membrane-derived protein radicals. With lytic doses of either oxidant, protein (probably hemoglobin)-derived, nitrogen-centered radicals are observed. Unlike the red blood cells, treatment of monocytes and macrophages with HOCl gives significant radical formation only under conditions where cell lysis occurs concurrently. These radicals are nitrogen-centered, cell-protein-derived species and have parameters identical to those detected with red blood cells and HOBr. Exposure of these cells to HOBr did not give detectable radicals. Overall these experiments demonstrate that HOCl and HOBr react with different selectivity with cellular targets, and that this can result in radical formation. This radical generation can precede, and may play a role in, cell lysis.     

Radical scavenging properties of genistein

The reactivity of genistein toward reactive radical species has been investigated by means of pulse radiolysis. The values of rate constants, respectively 2.3 x 10(10) M(-1)s(-1) and 1.3 x 10(10) M(-1)s(-1) for the reaction with hydroxyl radical at pH 8.3 and 3.0, are close to diffusion limit indicating that genistein is a potent hydroxyl radical scavenger. The reactivity of genistein towards one-electron oxidants has also been investigated. The rate constants k = 4.6 x 10(9) M(-1)s(-1) (pH 8.3) and 6.7 x 10(8) M(-1)s(-1) (pH 7.6) have been determined for the reaction of genistein with *N3 and Br2*- radicals, respectively. For both oxidants the rate constants at pH 3 does not exceed 10(8) M(-1)s(-1). The differences in reactivity of genistein towards the oxidants at different acidity of the solution have been assumed to arise from the acid-base equilibria of genistein. The dissociation constants for genistein (pKa: 7.2, 10.0, and 13.1) have been evaluated spectroscopically. The influence of acid-base equilibria on bond dissociation energy and ionization potential for genistein has also been investigated by means of DFT calculations. It has been concluded on the basis of these calculations that monoanionic form of genistein existing at physiological pH is more powerful radical scavenger than the neutral molecule.     

Kinetic properties of Cu,Zn-superoxide dismutase as a function of metal content--order restored

In a recent publication (Michel et al. Arch. Biochem. Biophys. 439:234-240; 2005) the authors argued that the catalytic rate constant, k(cat), for wild-type Cu,Zn-superoxide dismutase (Cu,Zn-SOD), determined previously by pulse radiolysis, was overestimated due to contamination with excess copper. They reported that addition of 0.1 mM EDTA to a sample that already contained excess copper did not remove spurious activity, which is incompatible with well-known stability constants of copper complexes and contradicts previous observations. In the present study we verified that the addition of EDTA eliminates completely the effect of excess copper on the decomposition rate of O2*- in the presence of Cu,Zn-SOD. We determined that k(cat) = (2.82 +/- 0.02) x 10(9) M(-1) s(-1) at low ionic strength (2 < I < 15 mM) and (1.30 +/- 0.02) x 10(9) M(-1) s(-1) in the presence of 50 mM phosphate at pH 7.8 (I = approximately 150 mM), which are about twice higher than those reported by Michel et al. We also determined k(cat) by the cytochrome c assay and demonstrated the correlation between these direct and indirect assays. The phenotypic deficits imposed by deletion of SODs, and the oxygen dependence of these deficits, have repeatedly demonstrated that the several SODs do in fact, as well as is theory, provide an important protection against that facet of oxidative stress imposed by O2*-.     

Dissociation of DNA double strand by hypohalous acids

Abstract

Calf thymus DNA was treated with authentic HOCl, and hypohalous acid-generating systems. This caused a decrease in fluorescence of ethidium-DNA complexes when ethidium bromide was subsequently added to the DNA. The fluorescence continued to decrease up to 30 min after adding HOCl. Loss in fluorescence was proportional to the concentration of HOCl and was complete when a 3-fold excess of HOCl was added to the DNA. No significant decrease in the fluorescence was observed when the chlorination was carried out in the presence of a concentration of monochlorodimedone (MCD) equivalent to that of HOCl. MCD is known to react stoichiometrically with HOCl. The decrease in fluorescence was completely inhibited by H₂O₂, ascorbate and glutathione (GSH). We have estimated the rate constant for the reaction of HOCl with H₂O₂ to be 1–2×10⁵ M^-1s^-1. When compared with authentic HOCl, HOCl-generating systems (Cl^-+H₂O₂+MPO or chloroperoxidase) were found to be inefficient in damaging DNA. This result most likely arises because the rate constant for reaction of HOCl with H₂O₂ is about 1000-fold faster than that for the reaction with DNA. HOBr and HOI generating systems also had a limited ability to damage DNA. We conclude that good chlorine acceptors and antioxidants protect DNA from hypohalous acid-induced oxidative damage.     

Generation of reactive oxygen species by photolysis of the ruthenium(II) complex Ru(NH3)5(pyrazine)2+ in oxygenated solution.

Metal-to-ligand charge transfer photolysis of the ruthenium(II) pyrazine complex Ru(NH3)5pz2+ (I) in pH 7.4 oxygenated phosphate buffer solution generates the Ru(III) analog Ru(NH3)5pz3+ plus the reactive oxygen species singlet oxygen and superoxide. Based on the very short MLCT lifetime (re-measured as approximately 250 ps in D2O) of I* and the quantum yield for singlet oxygen formation (0.01 for aerated D2O) the rate constant for oxygen quenching of I* was calculated to be approximately (3+/-1)x10(10) M-1 s-1.     

The mechanism of iron release from the transferrin-receptor 1 adduct

We report the determination in cell-free assays of the mechanism of iron release from the N-lobe and C-lobe of human serum transferrin in interaction with intact transferrin receptor 1 at 4.3< or =pH< or =6.5. Iron is first released from the N-lobe in the tens of milliseconds range and then from the C-lobe in the hundreds of seconds range. In both cases, iron loss is rate-controlled by slow proton transfers, rate constant for the N-lobe k(1)=1.20(+/-0.05)x10(6)M(-1)s(-1) and for the C-lobe k(2)=1.6(+/-0.1)x10(3)M(-1)s(-1). This iron loss is subsequent to a fast proton-driven decarbonation and is followed by two proton gains, (pK(1a))/2=5.28 per proton for the N-lobe and (pK(2a))/2=5.10 per proton for the C-lobe. Under similar experimental conditions, iron loss is about 17-fold faster from the N-lobe and is at least 200-fold faster from the C-lobe when compared to holotransferrin in the absence of receptor 1. After iron release, the apotransferrin-receptor adduct undergoes a slow partial dissociation controlled by a change in the conformation of the receptor; rate constant k(3)=1.7(+/-0.1)x10(-3)s(-1). At endosomic pH, the final equilibrated state is attained in about 1000 s, after which the free apotransferrin, two prototropic species of the acidic form of the receptor and apotransferrin interacting with the receptor coexist simultaneously. However, since recycling of the vesicle containing the receptor to the cell surface takes a few minutes, the major part of transferrin will still be forwarded to the biological fluid in the form of the apotransferrin-receptor protein-protein adduct.     

Comparison of low-density lipoprotein modification by myeloperoxidase-derived hypochlorous and hypobromous acids.

Myeloperoxidase (MPO), a heme enzyme secreted by activated phagocytes, catalyzes the oxidation of halides to hypohalous acids. At plasma concentrations of halides, hypochlorous acid (HOCl) is the major strong oxidant produced. In contrast, the related enzyme eosinophil peroxidase preferentially generates hypobromous acid (HOBr). Since reagent and MPO-derived HOCl converts low-density lipoprotein (LDL) to a potentially atherogenic form, we investigated the effects of HOBr on LDL modification. Compared to HOCl, HOBr caused 2-3-fold greater oxidation of tryptophan and cysteine residues of the protein moiety (apoB) of LDL and 4-fold greater formation of fatty acid halohydrins from the lipids in LDL. In contrast, HOBr was 2-fold less reactive than HOCl with lysine residues and caused little formation of N-bromamines. Nevertheless, HOBr caused an equivalent increase in the relative electrophoretic mobility of LDL as HOCl, which was not reversed upon subsequent incubation with ascorbate, in contrast to the shift in mobility caused by HOCl. Similar apoB modifications were observed with HOBr generated by MPO/H(2)O(2)/Br(-). In the presence of equivalent concentrations of Cl(-) and Br(-), modifications of LDL by MPO resembled those seen in the presence of Br(-) alone. Interestingly, even at physiological concentrations of the two halides (100 mM Cl(-), 100 microM Br(-)), MPO utilized a portion of the Br(-) to oxidize apoB cysteine residues. MPO also utilized the pseudohalide thiocyanate to oxidize apoB cysteine residues. Our data show that even though HOBr has different reactivities than HOCl with apoB, it is able to alter the charge of LDL, converting it into a potentially atherogenic particle.