Dependence of H2O2 Formation by Rat Heart Mitochondria on Substrate Availability and Donor Age

We have examined the substrate specificity and inhibitor sensitivity of H₂O₂ formation by rat heart mitochondria. Active H₂O₂ production requires both a high fractional reduction of Complex I (indexed by NADH/NAD⁺ + NADH ratio) and a high membrane potential, . These conditions are achieved with supraphysiological concentrations of succinate. With physiological concentrations of NAD-linked substrates, rates of H₂O₂ formation are much lower (less than 0.1% of respiratory chain electron flux) but may be stimulated by the Complex III inhibitor antimycin A, but not by myxothiazol. Addition of Mn²⁺ to give 10 nmol/mg of mitochondrial protein enhances H₂O₂ production with all substrate combinations, possibly by repleting mitochondrial superoxide dismutase with this cation. Contrary to previously published work, no increased activity of H₂O₂ production was found with heart mitochondria from senescent (24 month) rats, relative to young adults (6 month).     

Homogeneous green catalysts for olefin oxidation by mono oxovanadium(V) complexes of hydrazone Schiff base ligands

Three mono oxovanadium(V) complexes of tridentate Schiff base ligands [VO(OMe)L¹] (1), [VO(OMe)L²] (2) and [VO(OMe)L³] (3) obtained by monocondensation of 3-hydroxy-2-naphthohydrazide and aromatic o-hydroxyaldehydes have been synthesized (H₂L¹ = (E)-3-hydroxy-N′-(2-hydroxy-3-methoxybenzylidene)-2-naphthohydrazide, H₂L² = (E)-3-hydroxy-N′-(2-hydroxybenzylidene)-2-naphthohydrazide and H₂L³ = (E)-N′-(5-bromo-2-hydroxybenzylidene)-3-hydroxy-2-naphthohydrazide). The complexes were characterized by spectroscopic methods in the solid state (IR) and in solution (UV-Vis, ¹H NMR). Single crystal X-ray analyses were performed with 1 and 2. The catalytic potential of these complexes has been tested for the oxidation of cyclooctene using H₂O₂ as the terminal oxidant. The effects of various parameters including the molar ratio of oxidant to substrate, the temperature, and the solvent have been studied. The catalyst 2 showed the most powerful catalytic activity in oxidation of various terminal, cyclic and phenyl substituted olefins. Excellent conversions have been obtained for the oxidation of cyclic and bicyclic olefins.     

Proton translocation during denitrification by a nitrifying-denitrifying Alcaligenes sp.

A heterotrophic nitrifying Alcaligenes sp. from soil was grown as a denitrifier on nitrate and subjected to oxidant pulse experiments to ascertain the apparent effeciencies of proton translocations during O₂ and nitrogen-oxide respirations. With endogenous substrate as the reducing agent the H⁺/2e^– ratios, extrapolated to zero amount of oxidant per pulse, were 9.4, 3.7, 4.3 and 3.5 for O₂, nitrate, nitrite and N₂O, respectively. The value for O₂ and those for the N-oxides are, respectively, somewhat larger and smaller than corresponding values for Paracoccus denitrificans. None of the three permeant ions employed with the Alcaligenes sp. (valinomycin-K⁺, thiocyanate and triphenylmethylphosphonium) was ideal for all purposes. Thiocyanate provided highest ratios for O₂ but abolished the oxidant pulse response for nitrate and N₂O. Valinomycin was slow to penetrate to the cytoplasmic membrane and relatively high concentrations were required for optimal performance. Triphenylmethylphosphonium enhanced passive proton permeability and diminished proton translocation at concentrations required to realize the maximal oxidant pulse response.     

Cytochrome c oxidase: the mechanistic significance of structural H+ in energy transduction

Changes in the bulk-phase concentration of O₂ and H⁺ associated with the reduction of O₂ to water are simultaneously determined in reactions catalyzed by fully reduced cytochrome c oxidase both isolated and embedded in liposomes. Consistent with the polyphasic kinetics of electron transfer through the oxidase, the time course of O₂ consumption and H⁺ translocation exhibit the following novel characteristics: (1) The uptake of scalar protons (H_m ⁺), the ejection of vectorial protons (H⁺ _v), and the consumption of O₂, all proceed in a kinetically polyphasic process. (2) During the first phase of the reaction the rates of O₂ uptake and H⁺ transfer are extremely fast and compatible with the rates of electron flow through the oxidase. (3) The K_m of the oxidase for O₂ is close to 75 M, the same for O₂ consumption and scalar H⁺ uptake. The V_max of O₂ reduction to water in reactions catalyzed by the isolated enzyme is, at least, 0.5 × 10⁴ s^–1. (4) The extent of vectorial H⁺ ejection by cytochrome c oxidase embedded in liposomes is an exponential function dependent on both enzyme concentration and extent of O₂ consumption. (5) The H⁺/O stoichiometry of H⁺ ejection is a variable that may reach a maximum value of 4.0 only when the enzyme undergoes net oxidation at extremely high enzyme/O₂ molar ratios. It is postulated that the generation of useful energy at the level of cytochrome c oxidase depends not only on the number of molecules of O₂ reduced to water but also on the extent and state of reduction and/or protonation of the enzyme.     

Inhibition of peroxidase-catalyzed protein tyrosine nitration by antithyroid drugs and their analogues

In this paper, we describe the effect of some commonly used thiourea-based antithyroid drugs and their analogues on the peroxidase-catalyzed nitration reactions. The nitration of bovine serum albumin (BSA) and cytochrome c was studied using the antibody against 3-nitro-l-tyrosine. This study reveals that the thione-based antithyroid drugs effectively inhibit lactoperoxidase (LPO)-catalyzed nitration of BSA. These compounds show very weak inhibition towards the nitration of cytochrome c. Some of these compounds also inhibit myeloperoxidase (MPO)-catalyzed nitration of l-tyrosine. A structure-activity correlation study on the peroxidase-catalyzed nitration of l-tyrosine reveals that the presence of thione/selone moiety is important for the inhibition. Although the presence of a free N-H group adjacent to CS moiety is necessary for most of the thiones to inhibit the LPO-catalyzed nitration, the corresponding selones do not require the presence of any free N-H group for their activity. Furthermore, experiments with different concentrations of H₂O₂ suggest that the antithyroid drugs and related thiones inhibit the nitration reaction mainly by coordinating to the Fe(III)-center of the enzyme active site as previously proposed for the inhibition of peroxidase-catalyzed iodination. On the other hand, the selenium compounds inhibit the nitration by scavenging H₂O₂ without interacting with the enzyme active site. This assumption is based on the observations that catalase effectively inhibits tyrosine nitration by scavenging H₂O₂, which is one of the substrates for the nitration. In contrast, superoxide dismutase (SOD) does not alter the nitration reactions, indicating the absence of superoxide radical anion (O₂^-) during the peroxidase-catalyzed nitration reactions.     

Glyoxal oxidases: their nature and properties

H₂O₂ has been found to be required for the activity of the main microbial enzymes responsible for lignin oxidative cleavage, peroxidases. Along with other small radicals, it is implicated in the early attack of plant biomass by fungi. Among the few extracellular H₂O₂-generating enzymes known are the glyoxal oxidases (GLOX). GLOX is a copper-containing enzyme, sharing high similarity at the level of active site structure and chemistry with galactose oxidase. Genes encoding GLOX enzymes are widely distributed among wood-degrading fungi especially white-rot degraders, plant pathogenic and symbiotic fungi. GLOX has also been identified in plants. Although widely distributed, only few examples of characterized GLOX exist. The first characterized fungal GLOX was isolated from Phanerochaete chrysosporium. The GLOX from Utilago maydis has a role in filamentous growth and pathogenicity. More recently, two other glyoxal oxidases from the fungus Pycnoporus cinnabarinus were also characterized. In plants, GLOX from Vitis pseudoreticulata was found to be implicated in grapevine defence mechanisms. Fungal GLOX were found to be activated by peroxidases in vitro suggesting a synergistic and regulatory relationship between these enzymes. The substrates oxidized by GLOX are mainly aldehydes generated during lignin and carbohydrates degradation. The reactions catalysed by this enzyme such as the oxidation of toxic molecules and the production of valuable compounds (organic acids) makes GLOX a promising target for biotechnological applications. This aspect on GLOX remains new and needs to be investigated.     

Human Indoleamine 2,3-Dioxygenase Is a Catalyst of Physiological Heme Peroxidase Reactions: IMPLICATIONS FOR THE INHIBITION OF DIOXYGENASE ACTIVITY BY HYDROGEN PEROXIDE*

The heme enzyme indoleamine 2,3-dioxygenase (IDO) is a key regulator of immune responses through catalyzing l-tryptophan (l-Trp) oxidation. Here, we show that hydrogen peroxide (H₂O₂) activates the peroxidase function of IDO to induce protein oxidation and inhibit dioxygenase activity. Exposure of IDO-expressing cells or recombinant human IDO (rIDO) to H₂O₂ inhibited dioxygenase activity in a manner abrogated by l-Trp. Dioxygenase inhibition correlated with IDO-catalyzed H₂O₂ consumption, compound I-mediated formation of protein-centered radicals, altered protein secondary structure, and opening of the distal heme pocket to promote nonproductive substrate binding; these changes were inhibited by l-Trp, the heme ligand cyanide, or free radical scavengers. Protection by l-Trp coincided with its oxidation into oxindolylalanine and kynurenine and the formation of a compound II-type ferryl-oxo heme. Physiological peroxidase substrates, ascorbate or tyrosine, enhanced rIDO-mediated H₂O₂ consumption and attenuated H₂O₂-induced protein oxidation and dioxygenase inhibition. In the presence of H₂O₂, rIDO catalytically consumed nitric oxide (NO) and utilized nitrite to promote 3-nitrotyrosine formation on IDO. The promotion of H₂O₂ consumption by peroxidase substrates, NO consumption, and IDO nitration was inhibited by l-Trp. This study identifies IDO as a heme peroxidase that, in the absence of substrates, self-inactivates dioxygenase activity via compound I-initiated protein oxidation. l-Trp protects against dioxygenase inactivation by reacting with compound I and retarding compound II reduction to suppress peroxidase turnover. Peroxidase-mediated dioxygenase inactivation, NO consumption, or protein nitration may modulate the biological actions of IDO expressed in inflammatory tissues where the levels of H₂O₂ and NO are elevated and l-Trp is low.     

Role of hydroxyl radical in the oxidant H2O2-mediated Ca2+ release from pulmonary smooth muscle mitochondria

We sought to investigate the mechanism(s) by which the oxidant H₂O₂ stimulates Ca²⁺ release from mitochondria of bovine pulmonary vascular smooth muscle tissue and to test the hypothesis that hydroxyl radical is involved in this phenomenon. Treatment of the smooth muscle tissue with 1 mM H₂O₂ dramatically stimulated hydroxyl radical generation as measured by methane (CH₄) production by GLC using dimethylsulfoxide (DMSO) as the substrate. Pretreatment of the mitochondria with the hydroxyl radical scavanger dimethylthiourea (DMTU) prevented the increase in CH₄ production caused by H₂O₂. In the absence of EGTA, H₂O₂ caused stimulation of Ca²⁺ release from mitochondria occurred with a lag time of about 4 min. Addition of EGTA to Ca²⁺ loaded mitochondria resulted an immediate loss of Ca²⁺ and that has been found to be augmented by H₂O₂. The release of Ca₂₊ by H₂O₂ did not appear to occur with concommitant increase in sucrose entry into, K⁺ release from, and swelling of mitochondria when the Ca²⁺ cycling was prevented by EGTA. These observations suggested that H₂O₂-mediated Ca²⁺ release from bovine pulmonary vascular smooth muscle tissue mitochondria occurred (i) through the involvement of hydroxyl radical; (ii) via specific pathway(s); and (iii) did not appear to happen primarily via nonspecific pore formation.Abbreviations H₂O₂ hydrogen peroxide - OH· hydroxyl radical - t-buOOH tert-butyl hydroperoxide - CH₄ methane - GLC gas liquid chromatography - DMTU dimethylthiourea - EGTA ethylene glycol bis(-aminoethyl ether) - N Ntetraacetic acid - DMSO dimethyl sulfoxide - CH₄ methane - HBPS Hank's buffered physiological saline - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MOPS 3-(N-morpholino)propane sulfonic acid - Tris tris (hydroxymethyl aminomethane)     

Use of competitive dead-end inhibitors to determine the chemical mechanism of action of yeast alcohol dehydrogenase

In this work, we have postulated a comprehensive and unified chemical mechanism of action for yeast alcohol dehydrogenase (EC 1.1.1.1, constitutive, cytoplasmic), isolated from Saccharomyces cerevisiae. The chemical mechanism of yeast enzyme is based on the integrity of the proton relay system: His-51....NAD⁺....Thr-48....R.CH₂OH(H₂>O)....Zn$++,\; stretching\; from\; His-51\; on\; the\; surface\; of\; enzyme\; to\; the\; active\; site\; zinc\; atom\; in\; the\; substrate-binding\; site\; of\; enzyme.\; Further,\; it\; is\; based\; on\; extensive\; studies\; of\; steady-state\; kinetic\; properties\; of\; enzyme\; which\; were\; published\; recently.\; In\; this\; study,\; we\; have\; reported\; the\; pH-dependence\; of\; dissociation\; constants\; for\; several\; competitive\; dead-end\; inhibitors\; of\; yeast\; enzyme\; from\; their\; binary\; complexes\; with\; enzyme,\; or\; their\; ternary\; complexes\; with\; enzyme\; and\; NAD+or\; NADH;\; inhibitors\; include:\; pyrazole,\; acetamide,\; sodium\; azide,\; 2-fluoroethanol,\; and\; 2,2,2-trifluorethanol.\; The\; unified\; mechanism\; describes\; the\; structures\; of\; four\; dissociation\; forms\; of\; apoenzyme,\; two\; forms\; of\; the\; binary\; complex\; E.NAD+,\; three\; forms\; of\; the\; ternary\; complex\; E.NAD+.alcohol,\; two\; forms\; of\; the\; ternary\; complex\; E.NADH.aldehyde\; and\; three\; binary\; complexes\; E.NADH.\; Appropriate\; pK$_a values have been ascribed to protonation forms of most of the above mentioned complexes of yeast enzyme with coenzymes and substrates.     

Hybrid Mn-Peroxidase from the Ligninolytic Fungus Panus tigrinus 8/18. Isolation, Substrate Specificity, and Catalytic Cycle

Increased manganese concentration during submerged cultivation of the ligninolytic white rot fungus Panus tigrinus 8/18 on N-limited mineral medium resulted in the induction of Mn-peroxidase and laccase. The Mn-peroxidase was purified with the purity factor RZ (A ₄₀₆/A ₂₈₀) = 4.3. The purified enzyme catalyzed H₂O₂-dependent oxidation of phenol oxidase substrates (aromatic amines, 2,2"-azinobis-(3-ethylbenzthiazolinesulfonic acid), hydroquinone, 2,6-dimethoxyphenol) without Mn²⁺, which is not typical for the usual Mn-peroxidases. Guaiacol and 2,4,6-trichlorophenol were not oxidized in the absence of Mn²⁺. Study of absorption spectra of the intermediates of the catalytic cycle revealed that this peroxidase is able to complete the redox cycle, reducing one-electron oxidized intermediate (Compound II) by Mn²⁺, as well as by an organic substrate (hydroquinone). This means that the enzyme is a hybrid Mn-peroxidase, different from the common Mn-peroxidases from ligninolytic fungi.     

Effect of Fenton's pretreatment on cotton cellulosic substrates to enhance its enzymatic hydrolysis response

Fenton’s reagent that generates reactive hydroxyl radical species was evaluated for its effectiveness as a pretreatment agent on cotton cellulosic substrates to increase its susceptibility to cellulase enzyme. Response surface methodology was used to optimize four different process variables viz., time of reaction; substrate size and concentrations of Fe²⁺ and H₂O₂. Overall, the cellulose substrates treated at 0.5 mM concentration of Fe²⁺, 2% concentration of H₂O₂ for a reaction period of 48 h gave the highest enzyme activity as determined using the response surface methodology. Cellulose substrates with high aspect ratio recorded better enzyme response than that with low aspect ratio which is supported by copper number estimation. The cellulosic substrate prepared using a combination of optimized Fenton’s pretreatment conditions and/or enzyme hydrolysis were studied and characterized by atomic force microscopy and scanning electron microscopy. Additionally, degree of polymerization analysis gives further insight into the degradation during Fenton’s reaction.     

Reductive thiocyanolysis of tetraoxorhenate (VII): Synthesis, crystal structure, catalytic oxidation and kinetic studies of (PPh4)2[Re(NCS)6] and (PPh4)2[ReO(NCS)5]

Thiocyanate ions reduce perrhenate in aqueous acidic solution, and on addition of a suitable countercation (PPh₄Cl) afford (PPh₄)₂[Re(NCS)₆] (1) and (PPh₄)₂[ReO(NCS)₅] (2), which have been confirmed by X-ray crystallography. The kinetics of the above reaction has been studied. Both the complexes exhibit efficient and highly selective catalytic epoxidation ability in the presence of NaHCO₃ as a co-catalyst and competent catalytic properties in the selective oxidation of alcohols to the corresponding aldehydes or ketones in the presence of pyrazole as an additive and sulfides to sulfoxides and sulfones. H₂O₂ was used as the terminal oxidant in all the above-mentioned oxidation reactions.     

Generation of superoxide anion and hydrogen peroxide at the surface of plant cells

In addition to well-known cell wall peroxidases, there is now evidence for the presence of this enzyme at the plasma membrane of the plant cells (surface peroxidase). Both are able to catalyze, through a chain of reactions involving the superoxide anion, the oxidation of NADH to generate hydrogen peroxide. The latter is oxidized by other wall-bound peroxidases to convert cinnamoyl alcohols into radical forms, which, then polymerize to generate lignin. However, there are other enzymes at the surface of plasma membranes capable of generating hydrogen peroxide (cell wall polyamine oxidase), superoxide anion (plasma membrane Turbo reductase), or both (plasma membrane flavoprotein?). These enzymes utilize NAD(P)H as a substrate. The Turbo reductase and the flavoprotein catalyze the univalent reduction of Fe³⁺ and then of O₂ to produce Fe²⁺ and \(O_2^{\bar \cdot } \) , respectively. The superoxide anion, in the acidic environment of the cell wall, may then dismutate to H₂O₂. These superoxide anion- and hydrogen peroxide-generating systems are discussed in relation to their possible involvement in physiological and pathological processes in the apoplast of plant cells.     

Functional model for intradiol-cleaving catechol 1,2-dioxygenase: Synthesis, structure, spectra, and catalytic activity of iron(III) complexes with substituted salicylaldimine ligands

A series of mononuclear iron(III) complexes with containing phenolate donor of substituted-salicylaldimine based ligands [Fe(L¹)(TCC)] · CH₃OH (1), [Fe(L²)(TCC)] · CH₃OH (2), [Fe(L³)(TCC)] (3), and [Fe(L⁴)(TCC)] (4) have been prepared and studied as functional models for catechol dioxygenases (H₂TCC = tetrachlorocatechol, or HL¹ = N′-(salicylaldimine)-N,N-diethyldiethylenetriamine, HL² = N′-(5-Br-salicylaldimine)-N,N-diethyldiethylenetriamine, HL³ = N′-(4,6-dimethoxy-salycyl-aldimine)-N,N-diethyl-diethylenetriamine, HL⁴ = N′-(4-methoxy-salicylaldimine)-N,N-diethyl-diethylenetriamine). They are structural models for inhibitors of enzyme-substrate adducts from the reactions of catechol 1,2-dioxygenases. Complexes 1-4 were characterized by spectroscopic methods and X-ray crystal structural analysis. The coordination sphere of Fe(III) atom of 1-4 is distorted octahedral with N₃O₃ donor set from the ligand and the substrate TCC occupying cis position, and Fe(III) is in high-spin (S = 5/2) electronic ground state. The in situ prepared iron(III) complexes without TCC, [Fe(L¹)Cl₂], [Fe(L²)Cl₂], [Fe(L³)Cl₂], and [Fe(L⁴)Cl₂] are reactive towards intradiol cleavage of the 3,5-di-tert-butylcatechol (H₂DBC) in the presence of O₂ or air. The reaction rate of catechol 1,2-dioxygenase depends on the redox potential and acidity of iron(III) ions in complexes as well as the substituent effect of the ligands. We have identified the reaction products and proposed the mechanism of the reactions of these iron(III) complexes with H₂DBC with O₂.     

Synthesis of benzene ruthenium triazolato complexes by [3+2] cycloaddition reactions of activated alkynes and fumaronitrile to benzene ruthenium azido complexes

The dinuclear complex [(η⁶-C₆H₆)Ru(μ-N₃)Cl]₂ (1) is obtained by the reaction of [(η⁶-C₆H₆)RuCl₂]₂ with sodium azide in ethanol. The benzene ruthenium β-diketonato complexes of the general formula [(η⁶-C₆H₆)Ru(L∩L)Cl] {L∩L = O,O′-acac (2); O,O′-bzac (3); O,O′-dbzm (4)} are obtained in methanol by the reaction of [(η⁶-C₆H₆)RuCl₂]₂ with the corresponding β-diketonates. These complexes further react with sodium azide in ethanol to yield complexes of the type [(η⁶-C₆H₆)Ru(L∩L)N₃] [L∩L = O,O′-acac (5); L∩L = O,O′-bzac (6); L∩L = O,O′-dbzm (7)]. The complexes 5-7 are obtained as well by treating 1 with sodium salts of β-diketonates. These neutral benzene ruthenium azido complexes undergo [3+2] dipolar cycloaddition reaction with activated alkynes (MeO₂CCCCO₂Me, EtO₂CCCCO₂Et) or fumaronitrile (NCHCCHCN) to yield the corresponding benzene ruthenium triazolato complexes; [(η⁶-C₆H₆)Ru(O,O′-acac){N₃C₂(CO₂Me)₂}] (8), [(η⁶-C₆H₆)Ru(O,O′-acac){N₃C₂(CO₂Et)₂}] (9), [(η⁶-C₆H₆)Ru(O,O′-acac){N₃C₂HCN}] (10), [(η⁶-C₆H₆)Ru(O,O′-bzac){N₃C₂HCN}] (11) and [(η⁶-C₆H₆)Ru(O,O′-dbzm){N₃C₂HCN}] (12). These complexes are fully characterized on the basis of microanalyses, FT-IR and FT-NMR spectroscopy. The molecular structure of [(η⁶-C₆H₆)Ru(O,O′- acac){N₃C₂(CO₂C₂H₅)₂}] (9) is confirmed by single crystal X-ray diffraction study.     

Upstream reactive oxidative species (ROS) signals in exogenous oxidative stress-induced mitochondrial dysfunction

Exogenous oxidative stress induces cell death, but the upstream molecular mechanisms involved of the process remain relatively unknown. We determined the instant dynamic reactions of intracellular reactive oxygen species (ROS, including hydrogen peroxide (H₂O₂), superoxide radical (O₂⁻), and nitric oxide (NO)) in cells exposed to exogenous oxidative stress by using a confocal laser scanning microscope. Stimulation with extracellular H₂O₂ significantly increased the production of intracellular H₂O₂, O₂⁻, and NO (P < 0.01) through certain mechanisms. Increased levels of intracellular ROS resulted in mitochondrial dysfunction, involving the impairment of mitochondrial activity and the depolarization of mitochondrial membrane potential. Mitochondrial dysfunction significantly inhibited the proliferation of human hepatoblastoma G2 (HepG2) cells and resulted in mitochondrial cytochrome c (cyt c) release. The results indicate that upstream ROS signals play a potential role in exogenous oxidative stress-induced cell death through mitochondrial dysfunction and cyt c release.     

Substrate specificity and stereoselectivity of fatty alcohol oxidase from the yeast Candida maltosa

Summary In Candida maltosa and other alkene-utilizing yeasts a membrane-bound fatty alcohol oxidase (FAOD) is induced by growth on n-alkenes. The oxidation of 1-alkanols to the corresponding aldehydes is accompanied by the stoichiometric consumption of 1 mol O₂ and formation of 1 mol hydrogen peroxide (H₂O₂). The FAOD of C. maltosa shows a broad substrate specificity. It catalyses the oxidation of 1-alkanols (C₄ to C₂₂), with a maximal activity of 1.85 gmmol H₂O₂/ min × mg protein for 1-octanol, as well as the transformation of 2-alkanols (C₈ to C₁₆) to ketones. Other compounds as ,-alkenediols, -hydroxypalmitic acid, phenylalkanols and terpene alcohols are substrates for the enzyme, although mostly with decreased activities. The oxidation of the racemic 2-alkanols by the FAOD proceeds with very high stereoselectivity for the R(–)-enatiomers only, leaving the S(+)-2-alkanol untouched. Offprint requests to: S. Mauersberger     

An NADH coupled assay system for galactose oxidase.

A galactose oxidase (EC 1.1.3.9); NADH-peroxidase (EC 1.11.1.1) coupled assay system is used for the estimation of galactose oxidase activity. Spectrophotometric measurement of NADH consumption yields direct quantitative value of enzymic activity or can be used for the end-point determination of the amount of galactose oxidase substrate present in test solutions. Use of similar coupled systems is suggested for the assay of other H₂O₂-producing enzymes and their substrates.