Inhibition of Peroxidase-Catalyzed Oxidation of Chromogenic Substrates by Propyl Gallate and Its Polydisulfide

Peroxidase-catalyzed oxidation of 2,2′-azino-di-(3-ethyl-2,3-dihydrobenzthiazoline-6-sulfonate) (ABTS) was competitively inhibited by propyl gallate (PG) and its polydisulfide (PGPDS) at 20° C in 0.015 M phosphate-citrate buffer (pH 6.0). Under these conditions, the values of the inhibition constant (K _i ) were equal to 62 and 5.6 μM, respectively, for PG and PGPDS. The stoichiometric inhibition factor (f; the number of radicals extinguished per molecule of an inhibitor) equaled 2.0 and 14.7, respectively, for PG and PGPDS. Peroxidase-catalyzed oxidation of o-phenylenediamine was barely affected by PG or PGPDS. PGPDS may be used as a stop-reagent of peroxidase-catalyzed ABTS oxidation, whereas PG may serve as a calibrating inhibitor in test systems for measurement of total antioxidant activity (in human biological fluids, natural preparations, juices, wines, and other objects).__________Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 4, 2005, pp. 376–382.Original Russian Text Copyright © 2005 by Naumchik, Karasyova, Metelitza.     

Inhibition of peroxidase oxidation of aromatic amines by substituted phenols

Peroxidase-catalyzed oxidation of o-phenylene diamine (OPD) was competitively inhibited by trimethylhydroquinone (TMHQ), 4-tert-butylpyrocatechol (In5), and 4,6-di-tert-butyl-3-sulfanyl-1,2-dihydroxybenzene (In6). In6 was the most efficient inhibitor (Ki = 11 microM at 20 degrees C in 0.015 M phosphate-citrate buffer, pH 6.0). The effects of In5 and In6 were not preceded by periods of induction of OPD oxidation products (contrary to TMHQ). Peroxidase-catalyzed oxidation of tetramethylbenzidine (TMB) was non-competitively inhibited by In6 and 3-(2-hydroxyethylthio)-4,6-di-tert-butylpyrocatechol (In4), whereas o-aminophenol (OAP) acted as a mixed-type inhibitor. The effects of all three inhibitors were preceded by an induction period, during which TMB oxidation products were formed. Again, In6 was the most efficient inhibitor (Ki = 16 microM at 20 degrees C in 0.015 M phosphate-citrate buffer supplemented with 5% ethanol, pH 6.0). Judging by the characteristics of the inhibitors, taken in aggregate, it is advisable to use the pairs OPD-In5 and OPD-In6 in systems for testing the total antioxidant activity of biological fluids of humans.     

Initiation and inhibition of free-radical processes in biochemical peroxide systems: A review

The role of complexes containing oxygen or peroxide in monooxygenase systems and models thereof, as well as in peroxidase-and quasi-peroxidase-catalyzed processes, has been reviewed. Pathways of conversion of these intermediate complexes involving single-electron (radical) and two-electron (heterolytic) mechanisms are dealt with. Peroxidase-catalyzed co-oxidation of aromatic amines and phenols is analyzed; inhibition and activation of peroxidase-catalyzed reactions are characterized quantitatively. Oxidation of chromogenic substrates (ABTS, OPD, and TMB) in the presence of phenolic inhibitors or polydisulfides of substituted phenols is characterized by inhibition constants (K _i, μmol). Activation of peroxidase-catalyzed oxidation of the same substrates is characterized by the degree (coefficient) of activation (α, M^?1), which was determined for 2-aminothiazole, melamine, tetrazole, and its 5-substituted derivatives. Examples of applied use of peroxidase-catalyzed enzyme and model systems are given (oxidation of organic compounds, chemical analysis, enzyme immunoassay, tests for antioxidant activity of biological fluids).     

Characterization of peroxidase-catalyzed oxidation of chromogenic substrates by tetrazole and its 5-substituted derivatives

Peroxidase-catalyzed oxidation of 2,2-azino-di(3-ethyl-benzthiazolydine-6-sulfonic acid) (ABTS) and 3,3',5,5'-tetramethylbenzidine (TMB) is activated by tetrazole and its 5-substituted derivatives--5-amino-(AmT), 5-methyl- (MeT), 5-phenyl- (PhT), and 5-CF3- (CF3-T) tetrazoles. In phosphate-citrate or phosphate buffer (pH 6.4 or 7.2; 20 degrees C), the activating effect of tetrazoles on TMB and ABTS oxidation decreased in the series AmT > MeT > T > PhT > CF3-T and T > AmT > MeT > PhT, respectively. The (coefficient) degree of activation (alpha), expressed in M(-1), determined for both substrates and all activators, depended on substrate type, buffer nature, and pH (it increased as pH increased from 6.4 to 7.2). For TMB oxidation, good correlation between lgalpha and the Hammet constants sigma(meta) for m-substituents in the benzene series NH2, CH3, C6H5, and CF3 was found. It is suggested that AmT, MeT, and T can be used as activators of peroxidase-catalyzed oxidation of TMB and ABTS, as well as in designing peroxidase-based biosensors.     

Propyl gallate inhibition of iodide and tetramethylbenzidine oxidation catalyzed by human thyroid peroxidase

The kinetic characteristics (kcat, Km, and their ratio) for oxidation of iodide (I-) at 25 degrees C in 0.2 M acetate buffer, pH 5.2, and tetramethylbenzidine (TMB) at 20 degrees C in 0.05 M phosphate buffer, pH 6.0, with 10% DMF catalyzed by human thyroid peroxidase (HTP) and horseradish peroxidase (HRP) were determined. The catalytic activity of HRP in I- oxidation was about 20-fold higher than that of HTP. The kcat/Km ratio reflecting HTP efficiency was 35-fold higher in TMB oxidation than that in I- oxidation. Propyl gallate (PG) effectively inhibited all four peroxidase processes and its effects were characterized in terms of inhibition constants Ki and the inhibitor stoichiometric coefficient f. For both peroxidases, inhibition of I- oxidation by PG was characterized by mixed-type inhibition; Ki for HTP was 0.93 microM at 25 degrees C. However, in the case of TMB oxidation the mixed-type inhibition by PG was observed only with HTP (Ki = 3.9 microM at 20 degrees C), whereas for HRP it acted as a competitive inhibitor (Ki = 42 microM at 20 degrees C). A general scheme of inhibition of iodide peroxidation containing both enzymatic and non-enzymatic stages is proposed and discussed.     

Initiation and Inhibition of Free Radical Processes in H2O2–Metmyoglobin (Methemoglobin)–2,2"-Azino-bis-(3-Ethylbenzthiazoline-6-Sulfonic Acid) Systems

Rates of free radical initiation were determined at 20°C in 10 mM phosphate buffer (pH 7.4) in the systems metmyoglobin (methemoglobin)–H₂O₂ using 2,2"-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) as the diammonium salt (ABTS). The catalytic activity of MetMb was 2-3-fold higher than that of MetHb. The process can be described by the Michaelis–Menten equation, from which effective values of K _m and V _max were calculated. Comparative kinetic studies on the inhibition of ABTS oxidation were carried out using Trolox, propylgallate (PG), polydisulfide of gallic acid (poly(DSG)), polydisulfide of (2-amino-4-nitrophenol) (poly(ADSNP)), and its conjugate with human serum albumin (HSA–poly(ADSNP)). The inhibitors were characterized by inhibition constants K _i and stoichiometric inhibition coefficients f (the number of radicals terminated by a single molecule of inhibitor). The minimum K _i and the maximum f values were obtained for poly(DSG), and in the system of MetHb–H₂O₂–ABTS they were 0.08 M and 27.5, respectively. According to their antiradical activities, the inhibitors can be arranged as follows: poly(DSG) > poly(ADSNP) > PG > Trolox. PG, poly(DSG), poly(ADSNP), and its conjugate with HSA are suggested as calibrators, i.e., inhibition standards for evaluation of antioxidant status of biological fluids in possible test systems based on the free radical-generating pair MetMb–H₂O₂ with ABTS as the acceptor of the active radicals.     

Inhibition of Peroxidase-Catalyzed Oxidation of Aromatic Amines by Substituted Phenols

Peroxidase-catalyzed oxidation of o-phenylene diamine (OPD) was competitively inhibited by trimethylhydroquinone (TMHQ), 4-tert-butylpyrocatechol (InH5), and 4,6-di-tert-butyl-3-sulfanyl-1,2-dihydroxybenzene (InH6). InH6 was the most efficient inhibitor (K _i = 11 M at 20°C in 0.015 M phosphate–citrate buffer, pH 6.0). The effects of InH5 and InH6 were not preceded by periods of induction of OPD oxidation products (contrary to TMHQ). Peroxidase-catalyzed oxidation of tetramethylbenzidine (TMB) was noncompetitively inhibited by InH6 and 3-(2-hydroxyethylthio)-4,6-di-tert-butylpyrocatechol (InH4), whereas o-aminophenol acted as a mixed-type inhibitor. The effects of all three inhibitors were preceded by an induction period, during which TMB oxidation products were formed. Again, InH6 was the most efficient inhibitor (K _i = 16 M at 20°C in 0.015 M phosphate–citrate buffer supplemented with 5% ethanol, pH 6.0). Judging by the characteristics of the inhibitors taken in aggregate, it is advisable to use the pairs OPD–InH5 and OPD–InH6 in systems for testing the total antioxidant activity of human biological fluids.     

Doxorubicin inhibits oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) by a lactoperoxidase/H(2)O(2) system by reacting with ABTS-derived radical

The effect of doxorubicin on oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) by lactoperoxidase and hydrogen peroxide has been investigated. It was found that: (1) oxidation of ABTS to its radical cation (ABTS*(+)) is inhibited by doxorubicin as evidenced by its induction of a lag period, duration of which depends on doxorubicin concentration; (2) the inhibition is due to doxorubicin hydroquinone reducing the ABTS*(+) radical (stoichiometry 1: 1.8); (3) concomitant with the ABTS*(+) reduction is oxidation of doxorubicin; only when the doxorubicin concentration decreases to a near zero level, net oxidation of ABTS could be detected; (4) oxidation of doxorubicin leads to its degradation to 3-methoxysalicylic acid and 3-methoxyphthalic acid; (5) the efficacy of doxorubicin to quench ABTS*(+) is similar to the efficacy of p-hydroquinone, glutathione and Trolox C. These observations support the assertion that under certain conditions doxorubicin can function as an antioxidant. They also suggest that interaction of doxorubicin with oxidants may lead to its oxidative degradation.     

Antioxidant activity: what do we measure?

Inhibition of oxidation of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) by free radicals generated by decomposition of 2,2'-azobis(2-amidopropane) (ABAP) by antioxidants and biological material was studied. A correlation was found between the ability of various substances to delay the onset of ABTS oxidation and their rapid reduction of the ABTS+* cation radical, and between the ability to reduce the maximal rate of ABTS oxidation and slow reduction of ABTS+*. The length of the lag period of ABTS oxidation was found to be independent of ABTS concentration. Similar decrease of peroxynitrite-induced ABTS+* formation by antioxidants was observed when the antioxidants were added before and after peroxynitrite. All these findings indicate that the main effect of antioxidants in this system is reduction of ABTS+* and not prevention of its formation. Reduction of oxidation products rather than inhibition of their formation may be the predominant mode of action of antioxidants in various assays of antioxidant activity.     

Laccase chloride inhibition reduction by an anthraquinonic substrate

Due to their low substrate specificity, fungal laccases have a great potential in industrial applications, including the bioremediation of colored wastewaters from textile industry. However, the presence of halides in these effluents (up to 1M NaCl) which inhibit laccases is a drawback for bioremediation processes. In order to develop an efficient enzymatic remediation process for textile dye effluent, the possibility to reduce this halide inhibition is conditioned by a better understanding of the phenomenon. The present study gives a detailed account of the kinetics of chloride inhibition of both ABTS (a model substrate) and ABu62 (an anthraquinonic acid dye) oxidations catalyzed by Trametes versicolor laccase (LacIIIb). Chloride inhibition can be described by a mixed model for ABTS and a non-competitive model for ABu62 and both inhibitions are linear suggesting a single inhibitory site for chloride. Experiments were also conducted in presence of both substrates. An apparent activation of laccase was observed in the presence of ABu62 leading to an enhancement of the oxidation rate of ABTS. The extent of activation increased in the presence of chloride anions. Finally, for the first time to our knowledge, we evidenced that inhibition of ABTS oxidation by chloride can be reduced in the presence of ABu62.     

A mechanism for NaCl inhibition of Reactive Blue 19 decolorization and ABTS oxidation by laccase

Laccases produced by white rot fungi have been extensively evaluated for their potential to decolorize textile wastewaters which contain salts like sodium chloride and sodium sulfate. The effect of sodium chloride and sodium sulfate on Trametes versicolor laccase during the decolorization of an anthraquinone dye (Reactive Blue 19) and the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were evaluated by steady-state kinetic analysis. The results showed that, while sodium sulfate did not affect laccase activity, sodium chloride inhibited both ABTS oxidation and dye decolorization. However, the type of inhibition was substrate-dependent: it was hyperbolic, noncompetitive with ABTS and parabolic, noncompetitive with Reactive Blue 19. Furthermore, the results suggested that two chlorides may bind to laccase in the presence of the dye unlike recent inhibition models which suggest that there is only one inhibition site. This investigation is the first to provide evidence for and to propose a two-site model of laccase inhibition, providing new insight into NaCl inhibition of laccase. The proposed model is also useful to predict decolorization rates in the presence of sodium chloride and to determine operating conditions that will minimize inhibition.     

A serine proteinase inhibitor isolated from Tamarindus indica seeds and its effects on the release of human neutrophil elastase

Proteinaceous inhibitors with high inhibitory activities against human neutrophil elastase (HNE) were found in seeds of the Tamarind tree (Tamarindus indica). A serine proteinase inhibitor denoted PG50 was purified using ammonium sulphate and acetone precipitation followed by Sephacryl S-300 and Sephadex G-50 gel filtration chromatographies. Inhibitor PG50 showed a Mr of 14.9 K on Sephadex G-50 calibrated column and a Mr of 11.6 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PG50 had selective activity while cysteine proteinases (papain and bromelain) and serine proteinases (porcine pancreatic elastase and bovine chymotrypsin) were not inhibited, it was strongly effective against serine proteinases such as bovine trypsin and isolated human neutrophil elastase. The IC50 value was determined to be 55.96 microg.mL-1. PG50 showed neither cytotoxic nor haemolytic activity on human blood cells. After pre-incubation of PG50 with cytochalasin B, the exocytosis of elastase was initiated using PAF and fMLP. PG50 exhibited different inhibition on elastase release by PAF, at 44.6% and on release by fMLP, at 28.4%. These results showed that PG50 preferentially affected elastase release by PAF stimuli and this may indicate selective inhibition on PAF receptors.     

Age and renal prostaglandin inhibition during exercise and heat stress.

Aging is associated with a number of physiological changes that may cause the kidney to rely to a greater extent on vasodilatory PGs for normal functioning. Acute exercise has been shown to cause renal vasoconstriction that may be partially buffered by vasodilatory PGs. To determine the relative importance of renal PGs during exercise in older adults, we compared the renal effects of the PG inhibitor ibuprofen (1.2 g/day for 3 days) vs. a placebo control in a cohort of eight younger (24 +/- 2 yr) and eight older (64 +/- 2 yr) women during treadmill exercise ( approximately 57% maximal oxygen consumption) in the heat (36 degrees C). This over-the-counter dose of ibuprofen reduced renal PG (i.e., PGE2) excretion by 47% (P < 0. 05). Acute exercise in the heat caused dramatic decreases in glomerular filtration rate, renal blood flow, and sodium excretion in both age groups. PG inhibition was associated with greater decreases in urine production and free water clearance (P < 0.05). There were no drug-related declines in glomerular filtration rate or renal blood flow. We conclude that PG inhibition has only modest effects on renal function during exercise. Also, the lack of hemodynamic changes with PG inhibition indicates that healthy well-hydrated older women are not in a renal PG-dependent state.     

Inhibition of foetal pulmonary choline-phosphate cytidylyltransferase under conditions favouring protein phosphorylation.

Choline-phosphate cytidylyltransferase (EC 2.7.7.15) activity from 25- and 29-day-foetal rabbit lungs was inhibited in both the cytosolic and the microsomal fractions by preincubation with MgATP. The inhibition of the cytosolic enzyme was greater when measured with added phosphatidylglycerol (PG) than without (78-89% versus 50-55%), whereas the inhibition of the microsomal enzyme did not exhibit this distinction (66-72% versus 60-70%). When preincubated with the buffer alone, the cytosolic enzyme was activated to a greater extent by added PG than was the microsomal enzyme (13-14-fold versus 2-3-fold). However, after preincubation with MgATP, the cytosolic enzyme was activated to a smaller extent by added PG (3-6-fold). The inhibition of the enzyme by MgATP required a preincubation and was absent when ADP or AMP was substituted for ATP. Moreover, ATP analogues such as adenosine 5'-[beta, gamma-methylene]triphosphate and adenosine 5'-[gamma-thio]triphosphate also failed to inhibit the enzyme when substituted for ATP in the preincubation. The inhibition by MgATP was not affected by including cyclic AMP in the preincubation, but Ca2+ ions alone or plus diacylglycerol in the preincubation increased the inhibition slightly. The inhibition was abolished by including an inhibitor of cyclic-AMP-dependent protein kinase in the preincubation. These observations, taken collectively, point to the inhibition of foetal pulmonary cytidylyltransferase through the phosphorylation of a protein and suggest that this key enzyme in lung surfactant production may be regulated through this mechanism.     

Activation of peroxidase-catalyzed oxidation of chromogenic substrates by tetrazole and its 5-substituted derivatives

Peroxidase-catalyzed oxidation of 2,2-azino-di(3-ethyl-benzthiazolydine-6-sulfonic acid) (ABTS) and 3,3,5,5-tetramethylbenzidine (TMB) is activated by tetrazole and its 5-substituted derivatives—5-amino-(AmT), 5-methyl-(MeT), 5-phenyl-(PhT), and 5-CF₃-(CF₃-T) tetrazoles. In phosphate-citrate or phosphate buffer (pH 6.4 or 7.2; 20°C), the activating effect of tetrazoles on TMB and ABTS oxidation decreased in the series AmT > MeT > T > PhT > CF₃-T and T > AmT > MeT > PhT, respectively. The coefficient (degree) of activation (), expressed in M^–1, determined for both substrates and all activators, depended on substrate type, buffer nature, and pH (it increased as pH increased from 6.4 to 7.2). For TMB oxidation, good correlation between log and the Hammet constants _meta for m-substituents in the benzene series NH₂, CH₃, C₆H₅, and CF₃ was found. It is suggested that AmT, MeT, and T can be used as activators of peroxidase-catalyzed oxidation of TMB and ABTS in enzyme immunoassay and designing peroxidase-based biosensors.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 2, 2005, pp. 148–157.Original Russian Text Copyright © 2005 by Karasyova, Gaponik, Metelitza.     

Inhibition of cyclooxygenase-2 prevents inflammation-mediated preterm labor in the mouse

Prostaglandins (PGs) have proven important during parturition, but inhibition of PG production treating preterm labor (PTL) results in significant maternal and fetal side effects. We hypothesize that specific inhibition of either cyclooxygenase (COX)-1 or -2 may result in separation of therapeutic and toxic effects. We demonstrate that COX-2, but not COX-1, is induced during inflammation-mediated PTL caused by lipopolysaccharide (LPS) administration. A two- to threefold increase in uterine and ovarian PG concentrations coincides with this induction of COX-2. The COX-2-selective inhibitor SC-236 proved effective in stopping preterm delivery and the increases in PGs. The COX-1-selective inhibitor SC-560 also attenuated uterine and ovarian PG production after LPS but did not inhibit PTL as efficiently as SC-236. COX-1-deficient mice, which show delay in the onset of term labor, exhibited no delay in onset of PTL after LPS. These findings suggest that the mechanisms for initiation of inflammation-mediated PTL and term labor differ and that selective COX-2 inhibition may provide a means of stopping inflammation-induced PTL in humans.     

Phosphatidylglycerol Degradation is One Crucial Reason for the Decrease of its Concentration in Wheat Leaves under Phosphate Deprivation Stress

Phosphatidylglycerol (PG) is of crucial importance for the organization and function of thylakoid membranes. The reason for a decrease of PG concentration in plants under phosphate deprivation stress still remains unclear. By comparing PG concentration and PG hydrolase activity of wheat leaves at different developmental stages, we show that when the first leaves are fully developed, the PG concentration and PG hydrolase activity in phosphate-deficient plants were similar to those of the controls. With age, there was a significant decrease in PG concentration combined with an increase in PG hydrolase activity for phosphate-deficient plants. The close correlation between the decrease in PG concentration and the increase in PG hydrolases activities suggests that PG hydrolysis was the most important factor responsible for the decrease in its concentration. The main hydrolysis products of PG are phosphatidic acid (PA), diacylglycerol (DAG) and free fatty acid (FFA). The application of an inhibitor, n-butanol, which blocks the formation of PA via the PLD pathway, to the in vitro enzyme reaction mixture may restrict PA and DAG production. Neomycin sulfate, a PLC inhibitor, was shown to exhibit significant inhibition in DAG generation. These results suggest that both PLD and PLC were responsible for PG degradation in the leaves of phosphate-deficient wheat. The possible role of PLA activity for PG degradation is also discussed.     

Alterations of matrix- and cell-associated proteoglycans inhibit osteogenesis and growth response to fibroblast growth factor-2 in cultured rat mandibular condyle and calvaria

Matrix and cell surface proteoglycans (PGs) may play important roles in the control of cellular actions of heparan-binding growth factors such as fibroblast growth factor (FGF) during chondrogenesis and osteogenesis. In this study, we used 4-methylumbelliferyl-beta-d-xyloside, an inhibitor of PG synthesis, and sodium chlorate, a competitive inhibitor of glycoconjugate sulfation, to determine the functional consequences of alterations of PG metabolism on osteogenesis and on FGF actions in neonatal rat condyle and calvaria in vitro. Biochemical analysis showed that beta-d-xyloside (1 mM) or chlorate (15 mM) treatment for 1-8 days inhibited cellular PG synthesis by 60-80% in condyle and calvaria, as evaluated by [35S]sulfate incorporation. Histochemistry and immunohistochemistry showed that the inhibition of PG synthesis by beta-d-xyloside resulted in reduced incorporation of chondroitin sulfate into cartilage and bone matrix. This was associated with a 75% reduction in cell growth in condyle, determined by DNA synthesis, and in collagenous matrix synthesis in condyle and calvaria, evaluated by tritiated proline incorporation and type I collagen immunohistochemistry. Morphological and quantitative autoradiographic analyses also showed that inhibition of PG synthesis by beta-d-xyloside blocked bone matrix formation by perichondral progenitor cells in condyles and by osteoblasts in calvaria. In addition, alteration of PG metabolism blocked the mitogenic response to rhFGF-2 in calvaria. The data show that functional proteoglycans are essential for osteogenesis and for the growth response to FGF-2 during osteogenic differentiation in vitro.     

Chlororespiration and the process of carotenoid biosynthesis

The plastoquinone pool during dark adaptation is reduced by endogenous reductants and oxidized at the expense of molecular oxygen. We report here on the redox state of plastoquinone in darkness, using as an indicator the chlorophyll fluorescence kinetics of whole cells of a Chlamydomonas reinhardtii mutant strain lacking the cytochrome b(6)f complex. When algae were equilibrated with a mixture of air and argon at 1.45% air, plastoquinol oxidation was inhibited whereas mitochondrial respiration was not. Consequently, mitochondrial oxidases cannot be responsible for the oxygen consumption linked to plastoquinol oxidation. Plastoquinol oxidation in darkness turned out to be sensitive to n-propyl gallate (PG) and insensitive to salicylhydroxamic acid (SHAM), whereas mitochondrial respiration was sensitive to SHAM and PG. Thus, both PG treatment and partial anaerobiosis allow to draw a distinction between an inhibition of plastoquinol oxidation and an inhibition of mitochondrial respiration, indicating the presence of a plastoquinol:oxygen oxidoreductase. The possible identification of this oxidase with an oxidase involved in carotenoid biosynthesis is discussed in view of various experimental data.