The oxidation of oxyhaemoglobin by glyceraldehyde and other simple monosaccharides.

Glyceraldehyde and other simple monosaccharides oxidize oxyhaemoglobin to methaemoglobin in phosphate buffer at pH 7.4 and 37 degrees C, with the concomitant production of H2O2 and an alpha-oxo aldehyde derivative of the monosaccharide. Simple monosaccharides also reduce methaemoglobin to ferrohaemichromes (non-intact haemoglobin) at pH 7.4 and 37 degrees C. Carbonmonoxyhaemoglobin is unreactive towards oxidation by autoxidizing glyceraldehyde. Free-radical production from autoxidizing monosaccharides with haemoglobins was observed by the e.s.r. technique of spin trapping with the spin trap 5,5-dimethyl-l-pyrroline N-oxide. Hydroxyl and l-hydroxyalkyl radical production observed from monosaccharide autoxidation was quenched in the presence of oxyhaemoglobin and methaemoglobin. The haemoglobins appear to quench the free radicals by reaction with the free radicals and/or the ene-diol precursor of the free radical.     

Reactions of Adriamycin with haemoglobin. Superoxide dismutase indirectly inhibits reactions of the Adriamycin semiquinone.

The Adriamycin semiquinone produced by the reaction of xanthine oxidase and xanthine with Adriamycin has been shown to reduce both methaemoglobin and cytochrome c. In air, but not N2, both reactions were inhibited by superoxide dismutase. With cytochrome c, superoxide formed by the rapid reaction of the semiquinone with O2, was responsible for the reduction. However, even in air, methaemoglobin was reduced directly by the Adriamycin semiquinone. Superoxide dismutase inhibited this reaction by removing superoxide and hence the semiquinone by displacing the equilibrium: Semiquinone + O2 in equilibrium or formed from quinone + O2-. to the right. This ability to inhibit indirectly reactions of the semiquinone could have wider implications for the protection given by superoxide dismutase against the cytotoxicity of Adriamycin. Oxidation of haemoglobin by Adriamycin has been shown to be initiated by a reversible reaction between the drug and oxyhaemoglobin, producing methaemoglobin and the Adriamycin semiquinone. Reaction of the semiquinone with O2 gives superoxide and H2O2, which can also react with haemoglobin. Catalase, by preventing this reaction of H2O2, inhibits oxidation of oxyhaemoglobin. Superoxide dismutase, however, accelerates oxidation, by inhibiting the reaction of the semiquinone with methaemoglobin by the mechanism described above. Although superoxide dismutase has a detrimental effect on haemoglobin oxidation, it may protect the red cell against more damaging reactions of the Adriamycin semiquinone.     

The reaction of menadione with haemoglobin. Mechanism and effect of superoxide dismutase.

1. Menadione was found to react with both the haem groups and the beta-93 thiol groups of haemoglobin. 2. It oxidized the haem groups of oxyhaemoglobin, giving mainly methaemoglobin and a smaller amount of haemichrome. The reaction rate was decrease in the presence of catalase and markedly accelerated in the presence of superoxide dismutase. It is proposed that the overall reaction involves the initial reversible formation of methaemoglobin and the semiquinone, and that the effect of superoxide dismutase is to prevent the reverse reaction, by removing superoxide and hene O2-. E.s.r. evidence for the information of the semi-quinone and its reactions is presented. 3. The reaction of menadione with the beta-93 thiol groups of haemoglobin appeared to be similar to that with other thiols, forming the 3-thioether derivative of menadione, but it was also accompanied by reduction of methaemoglobin. This reduction was prevented by superoxide dismutase, but appeared to be caused by the semiquinone radical, which was produced as an intermediate. 4. Reduced glutathione functioned only to a limited extent as a scavenger of the menadione semiquinone. Its main reaction was directly with menadione to form the thioether. Ascorbate was a more efficient scavenger, and accelerated the oxidation of oxyhaemoglobin by menadione. 5. The significance of these findings in relation to menadione-induced erythrocyte haemolysis is discussed.     

Reactions involving superoxide and normal and unstable haemoglobins.

Superoxide ions (O2-) oxidized oxyhaemoglobin to methaemoglobin and reduced methaemoglobin to oxyhaemoglobin. The reactions of superoxide and H2O2 with oxyhaemoglobin or methaemoglobin and their inhibition by superoxide dismutase or catalase were used to detect the formation of superoxide or H2O2 on autoxidation of oxyhaemoglobin. The rate of autoxidation was decreased at about 35% in the presence of both enzymes. The copper-catalysed autoxidation of Hb (haemoglobin) was also shown to involve superoxide production. Superoxide was released on autoxidation of three unstable haemoglobins and isolated alpha and beta chains, at rates faster than with Hb A. Reactions of superoxide with Hb Christchurch and Hb Belfast were identical with those with Hb A, and occurred at the same rate. Hb Koln contrasted with the other haemoglobins in that the thiol groups of residue beta-93 as well as the haem groups reacted with superoxide. Haemichrome formation from methaemoglobin occurred very rapidly with Hb Christchurch and Hb Belfast, as well as the isolated chains, compared with Hb A. The process did not involve superoxide production or utilization. The relative importance of autoxidation and superoxide production compared with haemichrome formation in the haemolytic process associated with these abnormal haemoglobins and thalassaemia is considered.     

The rate of reaction of superoxide radical ion with oxyhaemoglobin and methaemoglobin.

Superoxide radical ions (O2-) produced by the radiolytic reduction of oxygenated formate solutions and by the xanthine oxidase-catalysed oxidation of xanthine were shown to oxidize the haem groups in oxyhaemoglobin and reduce those in methaemoglobin as in reactions (1) and (2): (see articles) Reaction (1) is suppressed by reaction (8) when [O2-]exceeds 10 muM, but consumes all the O2- generated in oxyhaemoglobin solutions when [oxyhaemoglobin] greater than 160 muM and [O2-]less than 1 nM at pH 7. The yield of reaction (2) is also maximal in methaemoglobin solutions under similar conditions, but less than one haem group is reduced per O2- radical. From studies of (a) the yield of reactions (1) and (2) at variable [haemoglobin] and rates of production of O2-, (b) their suppression by superoxide dismutase, and (c) equilibria observed with mixtures of oxyhaemoglobin and methaemoglobin, it is shown that k1/k2=0.7 +/- 0.2 and k1 = (4 +/- 1) X 10(3) M-1-S-1 At pH7, and k1 and k2 decrease with increasing pH. Concentrations and rate constants are expressed in terms of haem-group concentrations. Concentrations of superoxide dismutase observed in normal erythrocytes are sufficient to suppress reactions (1) and (2), and hence prevent the formation of excessive methaemoglobin.     

Haemichrome formation from haemoglobin subunits by hydrogen peroxide.

The effect of H2O2 on ferrous human haemoglobin subunits (alphash-, betash-, alphapmb- and betapmb-chains) was studied. These chains were easily transformed to haemichrome by the addition of H2O2 or H2O2-generating systems, including glucose oxidase (EC 1.1.3.4) AND XANTHINE OXIDASE (EC 1.2.3.2), and this was ascertained by e.p.r. measurements and by absorption spectra. The changes in these haemoglobin subunits were not inhibited by superoxide dismutase (EC 1.15.1.1), but were decreased by catalase (EC 1.11.1.6). The rate of oxidation of alphapmb-chains was higher than that of alphash-chains, and the rate of oxidation of betapmb-chains was higher than that of betash-chains. Haemichrome was demonstrated to be formed directly from these ferrous chains by the attack by H2O2, and this process did not involve formation of methaemoglobin. On the basis of these findings the kinetics of the reaction between the haemoglobin subunits and H2O2 was studied, and the pathological significance of H2O2 in disorders of erythrocytes such as thalassaemia was discussed.     

Ascorbate removes key precursors to oxidative damage by cell-free haemoglobin in vitro and in vivo

Haemoglobin initiates free radical chemistry. In particular, the interactions of peroxides with the ferric (met) species of haemoglobin generate two strong oxidants: ferryl iron and a protein-bound free radical. We have studied the endogenous defences to this reactive chemistry in a rabbit model following 20% exchange transfusion with cell-free haemoglobin stabilized in tetrameric form [via cross-linking with bis-(3,5-dibromosalicyl)fumarate]. The transfusate contained 95% oxyhaemoglobin, 5% methaemoglobin and 25 microM free iron. EPR spectroscopy revealed that the free iron in the transfusate was rendered redox inactive by rapid binding to transferrin. Methaemoglobin was reduced to oxyhaemoglobin by a slower process (t(1/2) = 1 h). No globin-bound free radicals were detected in the plasma. These redox defences could be fully attributed to a novel multifunctional role of plasma ascorbate in removing key precursors of oxidative damage. Ascorbate is able to effectively reduce plasma methaemoglobin, ferryl haemoglobin and globin radicals. The ascorbyl free radicals formed are efficiently re-reduced by the erythrocyte membrane-bound reductase (which itself uses intra-erythrocyte ascorbate as an electron donor). As well as relating to the toxicity of haemoglobin-based oxygen carriers, these findings have implications for situations where haem proteins exist outside the protective cell environment, e.g. haemolytic anaemias, subarachnoid haemorrhage, rhabdomyolysis.     

Mechanism of methaemoglobin breakdown by the lysine-specific gingipain of the periodontal pathogen Porphyromonas gingivalis

Abstract The R- and K-gingipain proteases of Porphyromonas gingivalis are involved in proteolysis of haemoglobin from which the defensive dimeric haem pigment is formed. Whilst oxyhaemoglobin is refractory towards K-gingipain, methaemoglobin is rapidly degraded. Ligation of methaemoglobin with N3-, which effectively blocks haem dissociation from the protein, prevented haemoglobin breakdown. Haem-free globin was rapidly degraded by K-gingipain. These data emphasise the need for haemoglobin oxidation which encourages haem dissociation and makes the haem-free globin susceptible to proteolytic attack.     

Formation of hydroxyl radicals from hydrogen peroxide in the presence of iron. Is haemoglobin a biological Fenton reagent?

The ability of oxyhaemoglobin and methaemoglobin to generate hydroxyl radicals (OH.) from H2O2 has been investigated using deoxyribose and phenylalanine as 'detector molecules' for OH.. An excess of H2O2 degrades methaemoglobin, releasing iron ions that react with H2O2 to form a species that appears to be OH.. Oxyhaemoglobin reacts with low concentrations of H2O2 to form a 'reactive species' that degrades deoxyribose but does not hydroxylate phenylalanine. This 'reactive species' is less amenable to scavenging by certain scavengers (salicylate, phenylalanine, arginine) than is OH., but it appears more reactive than OH. is to others (Hepes, urea). The ability of haemoglobin to generate not only this 'reactive species', but also OH. in the presence of H2O2 may account for the damaging effects of free haemoglobin in the brain, the eye, and at sites of inflammation.     

Conversion of oxyhaemoglobin into methaemoglobin by ferricytochrome b5.

Ferricytochrome b5 was found to convert oxyhaemoglobin into methaemoglobin under conditions previously found to be optimal for complex-formation between ferricytochrome b5 and methaemoglobin [Mauk & Mauk (1982) Biochemistry 21, 4730-4734]. As this reaction is completely inhibited by CO, it is proposed that oxyhaemoglobin is oxidized after O2 dissociation, as has been suggested for the oxidation of oxyhaemoglobin by inorganic complexes. From the present analysis, ferricytochrome b5 seems unlikely to contribute significantly to methaemoglobin formation in vivo. Nevertheless, this observation provides a relatively convenient means of investigating the mechanism by which these two proteins interact.     

Formation of bile pigments by coupled oxidation of cobalt-substituted haemoglobin and myoglobin.

Treatment of cobalt-substituted haemoglobin and myoglobin with ascorbate and molecular O2 (coupled oxidation) resulted in biliverdin formation from the cobalt(II) derivatives but not from the cobalt(III) derivatives. This was apparently due to the inability of ascorbate to reduce cobalt(III) haemoproteins. Isomer analysis of the biliverdins produced from coupled oxidation of cobalt(II) oxyhaemoglobin suggested that the orientation of the cobalt protoporphyrin IX in the haem pocket differed slightly from that of the haem in native haemoglobin.     

Ascorbate interacts with reduced glutathione to scavenge phenoxyl radicals in HL60 cells

We have compared the abilities of ascorbate and reduced glutathione (GSH) to act as intracellular free radical scavengers and protect cells against radical-mediated lipid peroxidation. Phenoxyl radicals were generated in HL60 cells, through the action of their myeloperoxidase, by adding H2O2 and phenol. Normally cultured cells, which contain no ascorbate; cells that had been preloaded with ascorbate; and those that had been depleted of GSH with buthionine sulfoximine were investigated. Generation of phenoxyl radicals resulted in the oxidation of ascorbate and GSH. Ascorbate loss was much greater in the absence of GSH, and adding glucose gave GSH-dependent protection against ascorbate loss. Ascorbate, or glucose metabolism, had little effect on the GSH loss. Glutathionyl radical formation was detected by spin trapping with DMPO in cells lacking ascorbate, and the signal was suppressed by ascorbate loading. Addition of phenol plus H2O2 to the cells caused lipid peroxidation, as measured with C11-BODIPY. Peroxidation was greatest in cells that lacked both ascorbate and GSH. Either scavenger alone gave substantial inhibition but optimal protection was seen with both present. These results indicate that GSH and ascorbate can each act as an intracellular radical scavenger and protect against lipid peroxidation. With both present, ascorbate is preferred and acts as the ultimate radical sink for phenoxyl or glutathionyl radicals. However, GSH is still consumed by metabolically recycling dehydroascorbate. Thus, recycling scavenging by ascorbate does not spare GSH, but it does enable the two antioxidants to provide more protection against lipid peroxidation than either alone.     

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. Effects of the hexose monophosphate shunt as mediated by glutathione and ascorbate.

Lipid peroxidation and haemoglobin degradation were the two extremes of a spectrum of oxidative damage in red cells exposed to t-butyl hydroperoxide. The exact position in this spectrum depended on the availability of glucose and the ligand state of haemoglobin. In red cells containing oxy- or carbonmono-oxy-haemoglobin, hexose monophosphate-shunt activity was mainly responsible for metabolism of t-butyl hydroperoxide; haem groups were the main scavengers in red cells containing methaemoglobin. Glutathione, via glutathione peroxidase, accounted for nearly all of the hydroperoxide metabolizing activity of the hexose monophosphate shunt. Glucose protection against lipid peroxidation was almost entirely mediated by glutathione, whereas glucose protection of haemoglobin was only partly mediated by glutathione. Physiological concentrations of intracellular or extracellular ascorbate had no effect on consumption of t-butyl hydroperoxide or oxidation of haemoglobin. Ascorbate was mainly involved in scavenging chain-propagating species involved in lipid peroxidation. The protective effect of intracellular ascorbate against lipid peroxidation was about 100% glucose-dependent and about 50% glutathione-dependent. Extracellular ascorbate functioned largely without a requirement for glucose metabolism, although some synergistic effects between extracellular ascorbate and glutathione were observed. Lipid peroxidation was not dependent on the rate or completion of t-butyl hydroperoxide consumption but rather on the route of consumption. Lipid peroxidation appears to depend on the balance between the presence of initiators of lipid peroxidation (oxyhaemoglobin and low concentrations of methaemoglobin) and terminators of lipid peroxidation (glutathione, ascorbate, high concentrations of methaemoglobin).     

Involvement of superoxide anion in the reaction mechanism of haemoglobin oxidation by nitrite.

The sigmoidal time course of haemoglobin oxidation by nitrite, involving an initial slow reaction accompanied by a subsequent rapid reaction, was extensively explored. The initial slow reaction was much prolonged by the addition of superoxide dismutase to the reaction mixture. On the other hand, in the presence of superoxide anion generated by xanthine oxidase systems, the slow phase disappeared and the reaction changed to first-order kinetics. The oxidation of intermediate haemoglobins [defined as haemoglobin tetramer in which different chains (alpha- or beta-) are in the ferric state and in the ferrous state] such as (alpha 2+ beta 3+)2 and (alpha 3+ beta 2+)2 also proceeded in a sigmoidal manner. Similar effects of superoxide anion on these reactions were observed. Since the intermediate haemoglobins such as (alpha 2+ beta 3+)2 and (alpha 3+ beta 2+)2 were found to be produced by the oxidation of haemoglobin by nitrite, the changes in oxyhaemoglobin, intermediate haemoglobins and methaemoglobin during the reaction were followed by isoelectric-focusing electrophoresis. The amounts of (alpha 2+ beta 3+)2 were larger than those of (alpha 3+ beta 2+)2 at the initial stages of the reaction, suggesting that there is a functional difference between alpha- and beta-chains in the oxyhaemoglobin tetramer. On the basis of these results, a reaction model of the haemoglobin oxidation by nitrite was tentatively proposed. The changes in oxyhaemoglobin, intermediate haemoglobins and methaemoglobin were well fitted to the simulation curves generated from the reaction model. Details of the derivation of the equations used for kinetic analysis have been deposited as Supplement SUP 50112 (5 pages) with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K. from whom copies may be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

Aniline hydroxylase activities of haemoglobin: Kinetics and mechanism

The mechanism of the aniline hydroxylase activity of methaemoglobin in a monooxygenase system consisting of NADH as electron donor, riboflavin, FAD, FMN or methylene blue as electron carrier and methaemoglobin as the terminal oxidase has been studied. Hydrogen peroxide is produced from oxygen in a methaemoglobin-independent process. 4-Aminophenol is subsequently produced peroxidatively by an NADH-dependent process; NADH prevents a further oxidation of 4-aminophenol in the presence of haemoglobin. In the absence of electron carrier, NADH slowly reduces haemoglobin and then oxyhaemoglobin reacts with aniline to give 4-aminophenol. In the absence of electron donor and electron carrier, oxyhaemoglobin and aniline give rise to the reversible production of 4-aminophenol.     

HmuY haemophore and gingipain proteases constitute a unique syntrophic system of haem acquisition by Porphyromonas gingivalis

Haem (iron protoporphyrin IX) is both an essential growth factor and virulence regulator for the periodontal pathogen Porphyromonas gingivalis, which acquires it mainly from haemoglobin via the sequential actions of the R- and K-specific gingipain proteases. The haem-binding lipoprotein haemophore HmuY and its cognate receptor HmuR of P. gingivalis, are responsible for capture and internalisation of haem. This study examined the role of the HmuY in acquisition of haem from haemoglobin and the cooperation between HmuY and gingipain proteases in this process. Using UV-visible spectroscopy and polyacrylamide gel electrophoresis, HmuY was demonstrated to wrest haem from immobilised methaemoglobin and deoxyhaemoglobin. Haem extraction from oxyhaemoglobin was facilitated after oxidation to methaemoglobin by pre-treatment with the P. gingivalis R-gingipain A (HRgpA). HmuY was also capable of scavenging haem from oxyhaemoglobin pre-treated with the K-gingipain (Kgp). This is the first demonstration of a haemophore working in conjunction with proteases to acquire haem from haemoglobin. In addition, HmuY was able to extract haem from methaemalbumin, and could bind haem, either free in solution or from methaemoglobin, even in the presence of serum albumin.     

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. The relative roles of haem- and glutathione-dependent decomposition of t-butyl hydroperoxide and membrane lipid hydroperoxides in lipid peroxidation and haemolysis

Red cells exposed to t-butyl hydroperoxide undergo lipid peroxidation, haemoglobin degradation and hexose monophosphate-shunt stimulation. By using the lipid-soluble antioxidant 2,6-di-t-butyl-p-cresol, the relative contributions of t-butyl hydroperoxide and membrane lipid hydroperoxides to oxidative haemoglobin changes and hexose monophosphate-shunt stimulation were determined. About 90% of the haemoglobin changes and all of the hexose monophosphate-shunt stimulation were caused by t-butyl hydroperoxide. The remainder of the haemoglobin changes appeared to be due to reactions between haemoglobin and lipid hydroperoxides generated during membrane peroxidation. After exposure of red cells to t-butyl hydroperoxide, no lipid hydroperoxides were detected iodimetrically, whether or not glucose was present in the incubation. Concentrations of 2,6-di-t-butyl-p-cresol, which almost totally suppressed lipid peroxidation, significantly inhibited haemoglobin binding to the membrane but had no significant effect on hexose monophosphate shunt stimulation, suggesting that lipid hydroperoxides had been decomposed by a reaction with haem or haem-protein and not enzymically via glutathione peroxidase. The mechanisms of lipid peroxidation and haemoglobin oxidation and the protective role of glucose were also investigated. In time-course studies of red cells containing oxyhaemoglobin, methaemoglobin or carbonmono-oxyhaemoglobin incubated without glucose and exposed to t-butyl hydroperoxide, haemoglobin oxidation paralleled both lipid peroxidation and t-butyl hydroperoxide consumption. Lipid peroxidation ceased when all t-butyl hydroperoxide was consumed, indicating that it was not autocatalytic and was driven by initiation events followed by rapid propagation and termination of chain reactions and rapid non-enzymic decomposition of lipid hydroperoxides. Carbonmono-oxyhaemoglobin and oxyhaemoglobin were good promoters of peroxidation, whereas methaemoglobin relatively spared the membrane from peroxidation. The protective influence of glucose metabolism on the time course of t-butyl hydroperoxide-induced changes was greatest in carbonmono-oxyhaemoglobin-containing red cells followed in order by oxyhaemoglobin- and methaemoglobin-containing red cells. This is the reverse order of the reactivity of the hydroperoxide with haemoglobin, which is greatest with methaemoglobin. In studies exposing red cells to a wide range of t-butyl hydroperoxide concentrations, haemoglobin oxidation and lipid peroxidation did not occur until the cellular glutathione had been oxidized. The amount of lipid peroxidation per increment in added t-butyl hydroperoxide was greatest in red cells containing carbonmono-oxyhaemoglobin, followed in order by oxyhaemoglobin and methaemoglobin. Red cells containing oxyhaemoglobin and carbonmono-oxyhaemoglobin and exposed to increasing concentrations of t-butyl hydroperoxide became increasingly resistant to lipid peroxidation as methaemoglobin accumulated, supporting a relatively protective role for methaemoglobin. In the presence of glucose, higher levels of t-butyl hydroperoxide were required to induce lipid peroxidation and haemoglobin oxidation compared with incubations without glucose. Carbonmono-oxyhaemoglobin-containing red cells exposed to the highest levels of t-butyl hydroperoxide underwent haemolysis after a critical level of lipid peroxidation was reached. Inhibition of lipid peroxidation by 2,6-di-t-butyl-p-cresol below this critical level prevented haemolysis. Oxidative membrane damage appeared to be a more important determinant of haemolysis in vitro than haemoglobin degradation. The effects of various antioxidants and free-radical scavengers on lipid peroxidation in red cells or in ghosts plus methaemoglobin exposed to t-butyl hydroperoxide suggested that red-cell haemoglobin decomposed the hydroperoxide by a homolytic scission mechanism to t-butoxyl radicals.     

Characterization of one- and two-electron oxidations of glutathione coupled with lactoperoxidase and thyroid peroxidase reactions

Glutathione (GSH) was oxidized to GSSG in the presence of H2O2, tyrosine, and peroxidase. During the GSH oxidation catalyzed by lactoperoxidase, O2 was consumed and the formation of glutathione free radical was confirmed by ESR of its 5,5'-dimethyl-1-pyrroline-N-oxide adduct. When lactoperoxidase was replaced by thyroid peroxidase in the reaction system, the consumption of O2 and the formation of the free radical became negligibly small. These results led us to conclude that, in the presence of H2O2 and tyrosine, lactoperoxidase and thyroid peroxidase caused the one-electron and two-electron oxidations of GSH, respectively. It was assumed that GSH is oxidized by primary oxidation products of tyrosine, which are phenoxyl free radicals in lactoperoxidase reactions and phenoxyl cations in thyroid peroxidase reactions. When tyrosine was replaced by diiodotyrosine or 2,6-dichlorophenol, the difference in the mechanism between lactoperoxidase and thyroid peroxidase disappeared and both caused the one-electron oxidation of GSH. Iodides also served as an effective mediator of GSH oxidation coupled with the peroxidase reactions. In this case the two peroxidases both caused the two-electron oxidation of GSH.     

Free radical generation and coupled thiol oxidation by lactoperoxidase/SCN-/H2O2.

The lactoperoxidase-catalyzed oxidation of glutathione (GSH) and thiocyanate (SCN-) was studied. Oxidation of SCN- was recorded by ultraviolet spectroscopy and by electron spin resonance (ESR). Consumption of GSH was measured by amperometric titration. One or two moles of GSH was oxidized per mole of H2O2 added, depending on the reaction conditions. Omission of SCN- prevented the oxidation of GSH. The oxidation of GSH required only catalytic amounts of SCN-, which was therefore recycled. Iodide (I-) could replace SCN-, while chloride or bromide were ineffective. The apparent Michaelis constant for SCN- was 17 microM. Oxidation of SCN- gave rise to two reactive intermediates, one stable and one unstable. The stable intermediate (-OSC. = N-(?)) decayed by a second-order reaction with a rate constant of 1.1 M-1 s-1. The decay of the unstable radical was very fast. The data (a) explain the short- and long-term antibacterial effects of lactoperoxidase-halide-H2O2 system, (b) point to possible deleterious effects due to glutathione depletion, (c) are of relevance for free radical diseases involving sulphur-centered free radicals, and (d) support previous observations on lipid peroxidation/halogenation in biological membranes, liposomes, and unsaturated fatty acids.     

Ascorbate-dependent recycling of the vitamin E homologue Trolox by dihydrolipoate and glutathione in murine skin homogenates

In the redox antioxidant network, dihydrolipoate can synergistically enhance the ascorbate-dependent recycling of vitamin E. Since the major endogenous thiol antioxidant in biological systems is glutathione (GSH) it was of interest to compare the effects of dihydrolipoate with GSH on ascorbate-dependent recycling of the water-soluble homologue of vitamin E, Trolox, by electron spin resonance (ESR). Trolox phenoxyl radicals were generated by a horseradish peroxidase (HRP)-hydrogen peroxide (H2O2) oxidation system. In the presence of dihydrolipoate, Trolox radicals were suppressed until both dihydrolipoate and endogenous levels of ascorbate in skin homogenates were consumed. Similar experiments made in the presence of GSH revealed that Trolox radicals reappeared immediately after ascorbate was depleted and that GSH was not able to drive the ascorbate-dependent Trolox recycling reaction. However, at higher concentrations GSH was able to increase ascorbate-mediated Trolox regeneration from the Trolox radical. ESR and spectrophotometric measurements demonstrated the ability of dihydrolipoate or GSH to react with dehydroascorbate, the two-electron oxidation product of ascorbate in this system. Dihydrolipoate regenerated greater amounts of ascorbate at a much faster rate than equivalent concentrations of GSH. Thus the marked difference between the rate and efficiency of ascorbate generation by dihydrolipoate as compared with GSH appears to account for the different kinetics by which these thiol antioxidants influence ascorbate-dependent Trolox recycling.