The Stoichiometry of Tyrosinase-Catalyzed Oxidation of 4-Hydroxyanisole

Oxygen utilisation during tyrosinase-catalysed oxidation of 4-hydroxyanisole was investigated using an electron spin resonance technique which employs quantitative changes in the characteristics of the electron spin resonance spectrum of the spin label 3-carbamoyl-2,5-dihydro-2,2,5-5-tetramethyl-1-H-pyridoyl-1-yloxy (CTPO) to follow changes in the oxygen concentration. Reaction mixtures containing mushroom tyrosinase (15 μg ml^-1) and differing initial concentrations of 4-hydroxyanisole in aerated phosphate buffer at pH 6.8 were incubated at room temperature. The ratio of utilisation of oxygen was found to be in approximately 1:1 molar ratio with the initial 4-hydroxyanisole concentration in the reaction mixture between 50 and 200 μmol/1 4-hydroxyanisole. The results are consistent with the stoichiometry of oxygen utilisation being accounted for by the oxidation of 4-hydroxyanisole to anisyl quinone.     

Adding talc microparticles to Aspergillus terreus ATCC 20542 preculture decreases fungal pellet size and improves lovastatin production

Changing fungal morphology with the use of morphological engineering techniques leads to improving the production of metabolites by filamentous fungi in the submerged culture. Adding mineral microparticles is one such simple method to change fungal pellet size. Here, it was studied for a lovastatin producer, Aspergillus terreus ATCC 20542. The experiments were conducted in shake flasks and 10 μm talc microparticles were added to the preculture. Intrapellet oxygen concentration profiles were determined by an oxygen microprobe. Talc microparticles caused a decrease of A. terreus pellets diameter from about 2000 to 900 μm, dependent on their concentration in the preculture. Smaller pellets produced more lovastatin, whose titre exceeded then 120 mg L^?1, utilising more lactose. The decrease in pellet size resulted in changes of oxygen concentration profiles in the pellets. The estimated critical pellet diameter, at which the non‐oxygenated zone was observed in the centre of the pellets, was 1700 μm. Smaller pellets were fully penetrated by oxygen. To conclude, facilitated diffusion of oxygen into the pellets of smaller diameter and their less dense structure made lactose utilisation by A. terreus more efficient, which ultimately increased lovastatin production in the runs with talc microparticles added, compared to the control runs.     

Melting studies on spin-labeled polyadenylic–polyuridylic complexes

Temperature-dependent conformational transitions of spin-labeled poly rA, spin-labeled poly rU and the two-stranded helical complexes consisting either of spin-labeled rA·poly rU or spin-labeled poly rU·poly rA have been measured by electron spin resonance spectrocopy. The polynucleotides were spin labeled with 4-(2-iodoacetamido)2,2,6,6-tetramethylpiperidinooxyl and the spin label to nucleotide base ratio was approximately 1:600. The relationship between the log of tumbling time τ and the reciprocal absolute temperature for the spin-labeled single and double-stranded polynucleotides is presented. An agreement between T_m^OD (optical density melting) and T_m^sp (spin melting) is found for the complexes, which strongly supports the conclusion that the same temperature-dependent structural changes are monitored with both techniques.     

Oxidation of tetrahydrobiopterin by peroxynitrite or oxoferryl species occurs by a radical pathway

The molecular mechanisms of tetrahydrobiopterin (BH₄) oxidation by peroxynitrite (ONOO^-) was studied using ultra-weak chemiluminescence, electron paramagnetic resonance (EPR) and UV-visible diodearray spectrophotometry, and compared to BH₄ oxidation by oxoferryl species produced by the myoglobin/hydrogen peroxide (Mb/H₂O₂) system. The oxidation of BH₄ by ONOO^- produced a weak chemiluminescence, which was altered by addition of 50 mM of the spin trap α-(4-pyridyl-1-oxide)-N-tert butylnitrone (POBN). EPR spin trapping demonstrated that the reaction occurred at least in part by a radical pathway. A mixture of two spectra composed by an intense six-line spectrum and a fleeting weak nine-line one was observed when using ONOO^-. Mb/H₂O₂ produced a short-living light emission that was suppressed by the addition of BH₄. Simultaneous addition of POBN, BH₄ and Mb/H₂O₂ produced the same six-line EPR spectrum, with a signal intensity depending on BH₄ concentration. Spectrophotometric studies confirmed the rapid disappearance of the characteristic peak of ONOO^- (302 nm) as well as substantial modifications of the initial BH₄ spectrum with both oxidant systems. These data demonstrated that BH₄ oxidation, either by ONOO^- or by Mb/H₂O₂, occurred with the production of activated species and by radical pathways.     

Tetranitromethane modification of photosystem 2

Inhibition of photosystem 2 by the peptide-modification reagent, tetranitromethane, has been investigated with spinach digitonin particles. In the presence of tetranitromethane, (1) the initial fluoresence yield is suppressed with a concomitant elimination of the variable component of fluorescence; (2) the optical absorption transient at 820 nm, attributed to P680⁺, is greatly attenuated; (3) diphenylcarbazide-supported photoreduction of dichlorophenol indophenol is abolished; and (4) electron spin resonance Signal 2f and Signal 2s are eliminated. These results are consistent with multiple sites of modification in photosystem 2 by tetranitromethane, and suggest further that this reagent can inhibit charge stabilization in the reaction center.Abbreviations D₁ electron donor to P680⁺ in oxygen-inhibited photosystem 2 preparations - DPIP 2,6-dichlorophenol indophenol - esr electron spin resonance - F_i initial chlorophyll a fluorescence yield - F_max maximum chlorophyll a fluorescence yield - F_v variable chlorophyll a fluorescence yield - FWHM full width at half maximum - Mes 2-(N-morpholino)ethanesulfonic acid - P680 primary electron donor chlorophyll of photosystem 2 - Ph pheophytin - PS 2-photosystem 2 - Q_a primary quinone electron acceptor - Q_b secondary quinone acceptor - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine - TNM tetranitromethane     

Tyrosinase Autoactivation and the Problem of the Lag Period

Evidence is presented for the binding of the quinone oxidation product of the monohydric phenol substrate, 4-hydroxyanisole, to mushroom tyrosinase. Column chromatography and SDS-PAGE separation showed labelling of the enzyme when incubated with ¹⁴C ring-labelled 4-hydroxyanisole. It is proposed that covalent binding to the enzyme and other proteins is through reaction of accessible nucleophilic groups, including thiols and amino groups, with the anisylquinone. This reductive addition enables the indirect generation of the catecholic substrate, which acts as an electron donor for the bicupric active site of met-tyrosinase and explains the lag kinetics of tyrosinase oxidation of non-cyclizing substrates. The effects of diluting the enzyme or the addition of amino acids on the lag period was consistent with a mechanism involving indirect generation of the dihydric phenol, which acts as the met-enzyme-recruiting substrate.     

Oxidation of NADH by plant mitochondria: Kinetics and effects of calcium ions

The kinetics of NADH oxidation by the outer membrane electron transport system of intact beetroot (Beta vulgaris L.) mitochondria were investigated. Very different values for V_max and the K_m for NADH were obtained when either antimycin A-insensitive NADH-cytochrome c activity (V_max= 31 ± 2.5 nmol cytochrome c (mg protein)^?1 min^?1; K_m= 3.1 ± 0.8 μM) or antimycin A-insensitive NADH-ferricyanide activity (V_max= 1.7 ± 0.7 μmol ferricyanide (mg protein)^?1 min^?1; K_m= 83 ± 20 μM) were measured. As ferricyanide is believed to accept electrons closer to the NADH binding site than cytochrome c, it was concluded that 83 ± 20 μM NADH represented a more accurate estimate of the binding affinity of the outer membrane dehydrogenase for NADH. The low K_m determined with NADH-cytochrome c activity may be due to a limitation in electron flow through the components of the outer membrane electron transport chain. The K_m for NADH of the externally-facing inner membrane NADH dehydrogenase of pea leaf (Pisum sativum L. cv. Massey Gem) mitochondria was 26.7 ± 4.3 μM when oxygen was the electron acceptor. At an NADH concentration at which the inner membrane dehydrogenase should predominate, the Ca²⁺ chelator, ethyleneglycol-(β-aminoethylether)-N,N,-tetraacetic acid (EGTA), inhibited the oxidation of NADH through to oxygen and to the ubiquinone-10 analogues, duroquinone and ubiquinone-1, but had no effect on the antimycin A-insensitive ferricyanide reduction. It is concluded that the site of action of Ca²⁺ involves the interaction of the enzyme with ubiquinone and not with NADH.     

In vitro study of the antioxidant properties of non steroidal anti-inflammatory drugs by chemiluminescence and electron spin resonance (ESR)

Objectives. To determine the antioxidant activities of nonsteroidal anti-inflammatory drugs (NSAIDS), we examined by chemiluminescence (CL) and electron spin resonance (ESR) their scavenging properties towards lipid peroxides, hypochlorous acid and peroxynitrite.

Methods. The antioxidant properties of nimesulide (NIM), 4-hydroxynimesulide (4-HONIM), aceclofenac (ACLO), 4-hydroxyaceclofenac (4-HOA-CLO), diclofenac (DICLO) and indomethacin (INDO) were tested on four different reactive oxygen species (ROS) generating systems: (I) phorbol-myristate acetate (PMA)-activated neutrophils, (II) Fe²⁺/ascorbate-induced lipid peroxidation, (III) HOCl-induced light emission, (IV) the kinetics of ONOO^- decomposition followed by spectrophotometry. ROS production was monitored by luminol-enhanced CL or by ESR using two different spin traps.

Results. At 10 μM, ACLO, NIM, 4-HONIM, 4-HOA-CLO, and DICLO decreased luminol-enhanced CL generated by PMA-activated neutrophils. Inversely, INDO increased the luminol enhanced CL. Interestingly, hydroxylated metabolites were more potent antioxidants than the parent drugs. Furthermore, all drugs tested, excepted ACLO, lowered lipid peroxidation induced by Fe²⁺/ascorbate system. ACLO and DICLO, even at the highest concentration tested (100 μM), did not significantly lower HOCl induced CL, whereas the other drugs were potent scavengers. Finally, all the NSAIDS accelerated decomposition of ONOO^-, suggesting a potential capacity of the molecules to scavenge peroxynitrite.

Conclusion. The NSAIDs possess variable degrees of antioxidant activities, linked to their ability to react with HOCl, lipid peroxides or ONOO^-. These antioxidant activities could offer interesting targeted side-effects in the treatment of joint inflammatory diseases.     

Study of transplanted lichens with the use of physical methods: the case of <Emphasis Type="Italic">Xanthoria parietina</Emphasis> (L.) Th. Fr.

Electron paramagnetic resonance spectroscopy and inductively coupled plasma optical emission spectrometry were used to study the epiphytic lichen Xanthoria parietina (L.) Th. Fr. transplanted to natural and urbanized zones in the Moscow region. The combination of physical and lichenindication methods allowed us to determine periods as well as the magnitude of changes in the biophysical characteristics of the transplanted lichen in different environmental conditions. Electron paramagnetic resonance spectroscopy demonstrated that the number of paramagnetic centers in lichens transplanted to the natural zone did not change in comparison with initial value (2.6 ± 0.4)·10¹⁷ spin/mg) and remained stable after four weeks after transplantation. The number of paramagnetic centers in lichens transplanted to the urbanized zone increased by 1.1·10¹⁷ spin/mg by the end of the third week as compared with its initial value ((2.6 ± 0.4)·10¹⁷ spin/mg). Inductively coupled plasma optical emission spectrometry was used to measure concentration of iron, copper, manganese, magnesium, and zinc. We detected substantial changes in concentration of these elements during first four weeks after transplantation. For instance, concentration of copper in lichens transplanted to the urbanized zone increased from 7.5 ± 2.7 μg/g to 56.4 ± 8.6 μg/g by the end of the third week of the experiment.     

The Role of Reactive Oxygen Species in Mitochondrial Permeability Transition

We have provided evidence that mitochondrial membrane permeability transition induced by inorganic phosphate, uncouplers or prooxidants such as t-butyl hydroperoxide and diamide is caused by a Ca²⁺-stimulated production of reactive oxygen species (ROS) by the respiratory chain, at the level of the coenzyme Q. The ROS attack to membrane protein thiols produces cross-linkage reactions, that may open membrane pores upon Ca²⁺ binding. Studies with submitochondrial particles have demonstrated that the binding of Ca²⁺ to these particles (possibly to cardiolipin) induces lipid lateral phase separation detected by electron paramagnetic resonance experiments exploying stearic acids spin labels. This condition leads to a disorganization of respiratory chain components, favoring ROS production and consequent protein and lipid oxidation.     

Aerobic photoreactivity of synthetic eumelanins and pheomelanins: generation of singlet oxygen and superoxide anion

In this work, we examined photoreactivity of synthetic eumelanins, formed by autooxidation of DOPA, or enzymatic oxidation of 5,6‐dihydroxyindole‐2‐carboxylic acid and synthetic pheomelanins obtained by enzymatic oxidation of 5‐S‐cysteinyldopa or 1:1 mixture of DOPA and cysteine. Electron paramagnetic resonance oximetry and spin trapping were used to measure oxygen consumption and formation of superoxide anion induced by irradiation of melanin with blue light, and time‐resolved near‐infrared luminescence was employed to determine the photoformation of singlet oxygen between 300 and 600 nm. Both superoxide anion and singlet oxygen were photogenerated by the synthetic melanins albeit with different efficiency. At 450‐nm, quantum yield of singlet oxygen was very low (~10^?4) but it strongly increased in the UV region. The melanins quenched singlet oxygen efficiently, indicating that photogeneration and quenching of singlet oxygen may play an important role in aerobic photochemistry of melanin pigments and could contribute to their photodegradation and photoaging.     

In situ analysis of oxygen consumption and diffusive transport in high‐temperature acidic iron‐oxide microbial mats

The role of dissolved oxygen as a principal electron acceptor for microbial metabolism was investigated within Fe(III)‐oxide microbial mats that form in acidic geothermal springs of Yellowstone National Park (USA). Specific goals of the study were to measure and model dissolved oxygen profiles within high‐temperature (65–75°C) acidic (pH = 2.7–3.8) Fe(III)‐oxide microbial mats, and correlate the abundance of aerobic, iron‐oxidizing Metallosphaera yellowstonensis organisms and mRNA gene expression levels to Fe(II)‐oxidizing habitats shown to consume oxygen. In situ oxygen microprofiles were obtained perpendicular to the direction of convective flow across the aqueous phase/Fe(III)‐oxide microbial mat interface using oxygen microsensors. Dissolved oxygen concentrations dropped from ～ 50–60 μM in the bulk‐fluid/mat surface to below detection (< 0.3 μM) at a depth of ～ 700 μm (～ 10% of the total mat depth). Net areal oxygen fluxes into the microbial mats were estimated to range from 1.4–1.6 × 10^?4 μmol cm^?2 s^?1. Dimensionless parameters were used to model dissolved oxygen profiles and establish that mass transfer rates limit the oxygen consumption. A zone of higher dissolved oxygen at the mat surface promotes Fe(III)‐oxide biomineralization, which was supported using molecular analysis of Metallosphaera yellowstonensis 16S rRNA gene copy numbers and mRNA expression of haem Cu oxidases (FoxA) associated with Fe(II)‐oxidation.     

Scavenging of reactive oxygen species by chlorophyllin: An ESR study

The antioxidant effects of chlorophyllin (CHL), a water-soluble analog of the green plant pigment chlorophyll, on different reactive oxygen species (ROS) were investigated by electron spin resonance (ESR) spectroscopy. As a standard, we have used the ability of CHL to scavenge the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. CHL inhibits the formation of 5,5-dimethyl-1-pyrroline-N-oxide adduct with hydroxyl radical (DMPO-OH adduct) generated by γ-radiation in a dose-dependent manner. At a concentration of 1 mM, CHL caused more than 90% inhibition of ESR signal intensity of this adduct. However, the results obtained with the Fenton reaction were different. We also found evidence for the inhibition of ¹O₂-dependent formation of the 2,2,6,6-tetramethyl-piperidine oxide (TEMPO) radical during photosensitization of methylene blue with visible light. CHL was also able to inhibit hydrogen peroxide induced oxidation of phenol red. The rate constant of the reaction of CHL with H₂O₂ was found to be 2.7×10⁶ M^-1s^-1. In conclusion, CHL has potent antioxidant ability involving scavenging of various physiologically important ROS.     

A rapid high-yield isolation method for nuclear envelope ghosts

A method is presented for the isolation of nuclear envelopes from isolated Tetrahymena macronuclei. In principle, nuclei are treated with DNase and RNase at low Ca²⁺/Mg²⁺ concentration followed by an extraction with 1 NaCl. The major advantages of this method are: (1) Unfragmented nuclear envelopes are obtained in the form of ghosts consisting of two juxtaposing nuclear membranes interrupted by pores as revealed by thin-section and freeze-etch electron microscopy. (2) The ghosts are obtained in high yield (60%) within a short period (1 h). (3) The nuclear envelopes largely retain their lipid composition. An average ghost contains about 96% of total phospholipids of an average nucleus. Nuclei and ghosts reveal an almost identical pattern of phospholipids and fatty acids as shown by thin-layer and gas-chromatography. (4) The lipids in the ghosts largely remain arranged in bilayers as probed by electron spin resonance using 5-doxylstearic acid as a spin label.     

Electron-transport chain and coupled oxidative phosphorylation in methanol-grown Paracoccus denitrificans

Methanol dehydrogenase of Paracoccus denitrificans was shown to be very similar to the enzyme of Pseudomonas sp, M. 27. The K _m value for methanol with excess activator (ammonium ions) is 35 M. The pH optimum for enzyme activity with 2,6-dichlorophe-nolindophenol as electronacceptor was at 9.0 A CO-binding type of cytochrome c was present only in cells grown with methanol as carbon and energy source.It has been shown that methanol-oxidation involves electron-transport via cytochrome c and an a-type cytochrome to oxygen. Antimycin A did not inhibit this electron transport and 90% inhibition was obtained by 375 M potassium cyanide. Electron transport from endogenous substrates is possible via cytochrome b and possibly cytochrome o to oxygen. Potassium cyanide inhibited 90% of the electron transport via this pathway at a concentration of 1.42 mM. Measurement of respiration-driven proton translocation proved that during oxidation of methanol to formaldehyde by oxygen one mole of adenosine triphosphate is synthesized in the site 3 region of the electron transport chain. The H⁺/O value found confirmed the H⁺/site ratio of 3–4 found in heterotrophic grown cells. During electron transport from endogenous substrates to oxygen there is a possible synthesis of 3 moles of adenosine triphosphate.In heterotrophically grown cells electron transfer to oxygen follows almost only the branch of the respiratory chain containing cytochrome o. In methanol-grown cells the pathway via the a-type cytochrome seems more important.Abbreviations DCPIP 2,6-dichlorophenolindophenol - PMS phenazine methosulphate - EPR electron paramagnetic resonance - S.D. standard deviation - ATP adenosine triphosphate     

EPR study of 17O nuclear hyperfine interaction in cobalt-oxyhemoglobin: conformation of bound oxygen.

The electron spin resonance spectra of cobalt-oxyhemoglobin, with oxygen enriched to 95 atom % with ¹⁷O, are broadened due to ¹⁷O nuclear hyperfine interaction with the unpaired electron spin and show at X-band and 7°K the presence of several well-resolved ¹⁷O satellites at low and high field extremes, all of which are absent in ¹⁶O cobaltoxyhemoglobin. The ¹⁷O EPR spectra are interpreted in terms of two nonequivalent hyperfine interaction constants of 65± 5 G and 93 ± 5 G for the two oxygen atoms. The observed broadening and hyperfine splittings in the EPR spectra indicate complete transfer of the unpaired spin to oxygen orbitals. The observed difference in the hyperfine coupling constants of the two oxygen atoms establishes an asymmetric linkage of the oxygen molecule to the metal, consistent with the Pauling model of oxyhemoglobin.     

Competition between redox mediator and oxygen in the microbial fuel cell

The maximal rates and effective constants of 2,6-dichlorphenolindophenol and oxygen reduction by bacterim Gluconobacter oxydans in bacterial fuel cells under different conditions were evaluated. In an open-circuit mode, the rate of 2,6-dichlorphenolindophenol reduction coupled with ethanol oxidation under oxygen and nirogen atmospheres were 1.0 and 1.1 μM s^–1 g^–1, respectively. In closed-circuit mode, these values were 0.4 and 0.44 μM s^–1 g^–1, respectively. The initial rate of mediator reduction with the use of membrane fractions of bacteria in oxygen and nitrogen atmospheres in open-circuit mode were 6.3 and 6.9 μM s^–1 g^–1, whereas these values in closed-circuit mode comprised 2.2 and 2.4 μM s^–1 g^–1, respectively. The oxygen reduction rates in the presence and absence of 2,6-dichlorphenolindophenol were 0.31 and 0.32 μM s^–1 g^–1, respectively. The data obtained in this work demonstrated independent electron transfer from bacterial redox centers to the mediator and the absence of competition between the redox mediator and oxygen. The results can make it possible to reduce costs of microbial fuel cells based on activity of acetic acid bacteria G. oxydans.     

AMMONIUM AND UV RADIATION STIMULATE THE ACCUMULATION OF MYCOSPORINE‐LIKE AMINO ACIDS IN PORPHYRA COLUMBINA (RHODOPHYTA) FROM PATAGONIA,ARGENTINA1

The combined effects of ammonium concentration and UV radiation on the red alga Porphyra columbina (Montagne) from the Patagonian coast (Chubut, Argentina) was determined using short‐term (less than a week) experimentation. Discs of P. columbina were incubated with three ammonium concentrations (0, 50, and 300 μM NH₄Cl) in anilluminated chamber (PAR=300 μmol photons·m^?2·s^?1, UVA=15 W·m^?2, UVB=0.7 W·m^?2) at 15°C. Algae incubated at 300 μM ammonium showed a significant increase (P<0.05) in the concentration of mycosporine‐like amino acids (MAAs) compared with the initial value or with the other ammonium treatments. The increase of MAAs was, however, a function of the quality of irradiance received, with a higher increase in samples exposed to UVA compared with UVB (29% and 5% increase, respectively). However, UVB radiation was more effective in inducing MAA synthesis per unit energy received by the algae. Samples exposed to PAR only had an intermediate increase in MAA concentration of 16%. Chl a concentration decreased through the incubation with the greatest decrease at high ammonium concentration. Phycobiliprotein (BP) decreased through time with the smallest decrease occurring at high ammonium concentration. Photoinhibition (as a decrease of optimal quantum yield) was significantly greater under nitrogen‐deprived conditions than that under replete ammonium levels. Maximal gross photosynthesis (GP_max), as oxygen evolution, and maximal electron transport rate (ETR_max), as chl fluorescence, increased with the ammonium concentration. Positive relationships between maximal GP or ETR and pigment ratios (BP/chl a and MAAs/chl a) and negative relationships with chl a concentration were found.     

Singlet oxygen scavenging activity of tocopherol and plastochromanol in Arabidopsis thaliana: relevance to photooxidative stress

In the present study, singlet oxygen (¹O₂) scavenging activity of tocopherol and plastochromanol was examined in tocopherol cyclase‐deficient mutant (vte1) of Arabidopsis thaliana lacking both tocopherol and plastochromanol. It is demonstrated here that suppression of tocopherol and plastochromanol synthesis in chloroplasts isolated from vte1 Arabidopsis plants enhanced ¹O₂ formation under high light illumination as monitored by electron paramagnetic resonance spin‐trapping spectroscopy. The exposure of vte1 Arabidopsis plants to high light resulted in the formation of secondary lipid peroxidation product malondialdehyde as determined by high‐pressure liquid chromatography. Furthermore, it is shown here that the imaging of ultra‐weak photon emission known to reflect oxidation of lipids was unambiguously higher in vte1 Arabidopsis plants. Our results indicate that tocopherol and plastochromanol act as efficient ¹O₂ scavengers and protect effectively lipids against photooxidative damage in Arabidopsis plants.     

Reaction of Trigonopsis variabilis d-amino acid oxidase with 2,6-dichloroindophenol: kinetic characterisation and development of an oxygen-independent assay of the enzyme activity

2,6-Dichloroindophenol (DCIP) is shown to be utilised efficiently as electron acceptor replacing dioxygen in the reaction of Trigonopsis variabilis d-amino acid oxidase (TvDAO) with d-methionine as the substrate. The specificity constant for DCIP reduction at 30 °C is one-twelfth that of oxygen conversion into hydrogen peroxide. Time course analysis of simultaneous consumption of DCIP and dioxygen, recorded on-line by absorption and non-invasive fluorescence quenching, respectively, pinpoints the preferential utilisation of dioxygen; and reveals a maximum DCIP conversion rate that is independent of the initial concentration of dioxygen. A robust direct assay of TvDAO activity has been developed that does not require anaerobic reaction conditions. It was down-scaled to microtitre plate format and overcomes practical limitations of other assays due to the low affinity of TvDAO for dioxygen (K_m ≈ 0.7 mmol L⁻¹).