Glucose oxidation catalyzed by liposomal glucose oxidase in the presence of catalase-containing liposomes

A catalase-containing liposome (CAL) was prepared and characterized in terms of stability during storage and catalysis of the decomposition of hydrogen peroxide (H2O2) that was initially added or produced in the oxidation of glucose catalyzed by the glucose oxidase-containing liposomes (GOL). The reactors used were a test tube and an external loop airlift bubble column as the static liquid and circulating liquid flow systems, respectively. The free catalase (CA) at low concentrations was unstable during storage at 4 degrees C as a result of dissociation of the tetrameric CA subunits. On the other hand, the deactivation of the CA activity in the CAL was depressed because of the high CA concentration in the CAL liposome. The CAL effectively catalyzed the repeated decompositions at 25 degrees C with 10 mM H2O2 added initially, whereas the free CA was significantly deactivated during the repeated reactions. The high stability of the CAL was attributed to the moderately depressed reactivity, which was essentially derived from the diffusion limitation of the CAL membrane to H2O2 in the liquid bulk. In the GOL-catalyzed prolonged oxidation of 10 mM glucose at 40 degrees C in the static liquid in a test tube, both the free CA and CAL could continuously catalyze the decomposition of H2O2 produced. This was because the glucose oxidation rate was small due to the limited reactivity of the GOL to glucose with its low permeability through the GOL membrane. In the glucose oxidation catalyzed by the GOL with the free CA or the CAL in the airlift, much larger oxidation rates were observed compared to those in the test tube because the permeability of the GOL membrane to glucose was increased in the gas-liquid two phase flow in the airlift. The GOL/CAL system in the airlift operated in an acidic condition, which was preferable to the GO activity, gave the largest oxidation rate with negligible accumulation of the H2O2 produced. On the other hand, the GOL/free CA system gave an oxidation rate smaller than that of the GOL/CAL system even under the acidic condition due to an unfavorable interaction of the free CA molecules with the GOL membranes leading to the decreased reactivity of the GOL.     

Identification by electron spin resonance of free radicals formed during the oxidation of 4-hydroxyanisole catalyzed by tyrosinase

We have observed the formation of free radicals during the oxidation of the melanocytotoxic agent 4-hydroxyanisole with the enzyme tyrosinase as a catalyst. The first free radical to form is identified as the 4-methoxy-1,2-benzosemiquinone radical anion. The peak concentration of this radical increases with tyrosinase concentration; a minimum concentration of 50 micrograms/ml of tyrosinase was needed to observe this radical. The peak concentration of this radical is independent of 4-hydroxyanisole concentration. This radical is produced by reverse dismutation of the primary product, 4-methoxy-1,2-benzoquinone and 4-methoxycatechol produced indirectly.     

Mechanism of hydrogen peroxide formation catalyzed by NADPH oxidase in thyroid plasma membrane

The thyroid plasma membrane contains a Ca2(+)-regulated NADPH-dependent H2O2 generating system which provides H2O2 for the thyroid peroxidase-catalyzed biosynthesis of thyroid hormones. The plasma membrane fraction contains a Ca2(+)-independent cytochrome c reductase activity which is not inhibited by superoxide dismutase. But it is not known whether H2O2 is produced directly from molecular oxygen (O2) or formed via dismutation of super-oxide anion (O2-). Indirect evidence from electron scavenger studies indicate that the H2O2 generating system does not liberate O2-, but studies using the modified peroxidase, diacetyldeuteroheme horseradish peroxidase, to detect O2- indicate that H2O2 is provided via the dismutation of O2-. The present results provide indirect evidence that the cytochrome c reductase activity is not a component of the NADPH-dependent H2O2 generator, since it was removed by washing the plasma membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid without affecting H2O2 generation. Spectral studies with diacetyldeuteroheme-substituted horseradish peroxidase showed that the thyroid NADPH-dependent H2O2 generator does not catalyze superoxide anion formation. The O2- adduct compound (compound III) was formed but was completely inhibited by catalase, indicating that the initial product was H2O2. The rate of NADPH oxidation also increased in the presence of diacetylheme peroxidase. This increase was blocked by catalase and was greatly enhanced by superoxide dismutase. The O2- adduct compound (compound III) was produced in the presence of NADPH when glucose-glucose oxidase (which does not produce O2-) was used as the H2O2 generator. NADPH oxidation occurred simultaneously and was enhanced by superoxide dismutase. We conclude that O2- formation occurs in the presence of an H2O2 generator, diacetylheme peroxidase and NADPH, but that it is not the primary product of the H2O2 generator. We suggest that O2- formation results from oxidation of NADPH, catalyzed by the diacetylheme peroxidase compound I, producing NADP degree, which in turn reacts with O2 to give O2-.     

Enzyme catalyzed formation of radicals from S-adenosylmethionine and inhibition of enzyme activity by the cleavage products

A large superfamily of enzymes have been identified that make use of radical intermediates derived by reductive cleavage of S-adenosylmethionine. The primary nature of the radical intermediates makes them highly reactive and potent oxidants. They are used to initiate biotransformations by hydrogen atom abstraction, a process that allows a particularly diverse range of substrates to be functionalized, including substrates with relatively inert chemical structures. In the first part of this review, we discuss the evidence supporting the mechanism of radical formation from S-adenosylmethionine. In the second part of the review, we examine the potential of reaction products arising from S-adenosylmethionine to cause product inhibition. The effects of this product inhibition on kinetic studies of 'radical S-adenosylmethionine' enzymes are discussed and strategies to overcome these issues are reviewed. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.     

Glycation promotes the formation of genotoxic aggregates in glucose oxidase

This study investigates the effect of pentose sugars (ribose and arabinose) on the structural and chemical modifications in glucose oxidase (GOD) as well as genotoxic potential of this modified form. An intermediate state of GOD was observed on day 12 of incubation having CD minima peaks at 222 and 208?nm, characteristic of α-helix and a few tertiary contacts with altered tryptophan environment and high ANS binding. All these features indicate the existence of molten globule state of the GOD with ribose and arabinose on day 12. GOD on day 15 of incubation forms β structures as revealed by CD and FTIR which may be due to its aggregation. Furthermore, GOD on day 15 showed a remarkable increase in Thioflavin T fluorescence at 485?nm. Comet assay of lymphocytes and plasmid nicking assay in presence of glycated GOD show DNA damage which confirmed the genotoxicity of advance glycated end products. Hence, our study suggests that glycated GOD results in the formation of aggregates and the advanced glycated end products, which are genotoxic in nature.     

Excited carbonyl formation in the combination and disproportionation of free radicals

The pyrolyisis of di-tert-butyl peroxyoxalate in the presence of para-substituted benzaldehydes produces almost quantitatively the corresponding p,p′-disubstituted benzils. The formation of these products is accompanied by chemiluminescence arising from excited triplets. From the quantum yield of excited triplet generation and the rate constants for the triplet photocleavage it is possible to obtain the change in Gibbs free energy associated with triplet formation. The values obtained are ?5.6, ?5.7 and ?8.1 kcal/mol for benzil, p,p′-dimethylbenzil and p,p′-dimethoxybenzil, respectively. The pyrolysis of di-tert-butyl peroxyoxalate in the presence of isopropanol or benzoin leads to the formation of acetone and benzil. These products are generated in disproportionation processes involving the α-hydroxy radical produced by hydrogen abstraction. The luminescence observed in these reactions constitutes the first experimental indication of excited species generation in the disproportionation of uncorrelated free radicals.     

Enzymatic formation of propylene bromohydrin from propylene by glucose oxidase and lactoperoxidase

The time course of the peroxidative bromination of propylene accompanied by in situ generation of hydrogen peroxide by glucose oxidase was examined to improve the productivity of propylene bromohydrin. To prevent the rapid inactivation of lactoperoxidase by excess hydrogen peroxide, it was effective to use lactoperoxidase in large excess as compared with glucose oxidase, and to raise the concentration of bromide ion. However, the rate of glucose consumption was lowered at high concentrations of bromide ion, and at higher mole fraction of oxygen as compared with propylene in the gas mixture. Therefore, it seemed that for the favorable production of bromohydrin there existed the optimal conditions for the concentration of bromide ion and for the composition of oxygen-propylene gas mixture. Such kinetic behaviors of the sequential enzymatic reactions were explained by a mechanism involving free hypobromous acid as a reactive intermediate. Furthermore, the stability of the coimmobilized enzymes with k-carrageenan gels was investigated in continuous operations. The half-life of the enzymes was ca. 60 h for the production of propylene bromohydrin.     

The spin-trapping of enzymatically and chemically catalyzed free radicals from indolic compounds

A nitrogen-centered free radical was spin-trapped from superoxide-catalyzed oxidation of indolic compounds, using the spin-trap phenyl-t-butyl-nitrone. The hyperfine splitting constants observed were aN = 13.9 G, a beta N = 3.6 G and a beta H = 2.3 G. Incubation of various indolic compounds with goat lung microsomes showed that only 3-methylindole was able to generate a free radical in the NADPH-dependent microsomal system, as tested by spin-trapping. The splitting constants were the same as those seen with superoxide incubations with 3-methylindole. The study demonstrates the generality of formation of a nitrogen-centered free radical from various indolic compounds. Enzymatic radical formation from 3-methylindole suggests a microsomal-activated free radical mechanism for the specificity of 3-methylindole-induced pulmonary toxicity.     

Free radicals and anticancer drug resistance: Oxygen free radicals in the mechanisms of drug cytotoxicity and resistance by certain tumors

Certain anticancer agents form free radical intermediates during enzymatic activation. Recent studies have indicated that free radicals generated from adriamycin and mitomycin C may play a critical role in their toxicity to human tumor cells. Furthermore, it is becoming increasingly apparent that reduced drug activation and or enhanced detoxification of reactive oxygen species may be related to the resistance to these anticancer agents by certain tumor cell lines. The purposes of this review are to summarize the evidence pointing toward the significance of free radicals formation in drug toxicity and to evaluate the role of decreased free radical formation and enhanced free radical scavenging and detoxification in the development of anticancer drug resistance by a spectrum of tumor cell types. Studies failing to support the participation of oxyradicals in the cytotoxicity and resistance of adriamycin are also discussed.     

Comparative kinetic study of D-glucose oxidation by ruthenium(III) compounds catalyzed by FAD-dependent glucose oxidase and PQQ-dependent glucose dehydrogenase

The comparative kinetic study of two glucose oxidizing enzymes, FAD-dependent glucose oxidase and PQQ-dependent glucose dehydrogenase, is presented in the artificial electron transfer mediator system based on ruthenium(III) compounds. It is demonstrated that FAD-dependent glucose oxidase and PQQ-dependent glucose dehydrogenase follow Michaelis kinetics in the D-glucose/ruthenium(III) system. PQQ-dependent glucose dehydrogenase is more active than FAD-dependent glucose oxidase in the process of D-glucose oxidation by ruthenium(III) compounds, this being due to the different catalytic mechanisms of these enzymes.     

Psoralen-photosensitized formation of free lipid radicals at 77 degrees K

It is shown by ESR method that 8-methoxypsoralen (8-MOP) under low temperature UV-irradiation has no influence on free radical formation of saturated fatty acids (FA). Under the same conditions the quantum yield of free radical photogeneration for unsaturated FA (oleate and limolenate) after addition of 8-MOP increased 7.5-15 times. The ESR spectrum of free radicals generated after interaction of 8-MOP with polyunsaturated FA, may be presented as superposition of signals-CH-(CH = CH)2-, -CH2CHCH2- and -CH2CH2. The molecular mechanism of photosensitized reactions is discussed.     

Glutamate induces formation of free radicals in rat brain synaptosomes

The influence of glutamate and agonists of its ionotropic receptors on free radical formation in rat brain synaptosomes was investigated using the fluorescent dye DCFDA. Glutamate at concentrations of 100 μM and 1 mM increased the production of reactive oxygen species. This phenomenon was eliminated by removing calcium from the incubation medium. Addition of NMDA (100 μM) or kainate (100 μM) to a suspension of synaptosomes also led to free radical formation. The influence of glutamate receptor agonists was blocked by the specific antagonists MK-801 and NBQX. Thus, activation of NMDA and AMPA/kainate receptors can lead to oxidative stress in neuronal presynaptic endings.     

Sulfur oxidation of free methionine by oxygen free radicals

The oxidation of free methionine to methionine sulfoxide by chemically or enzymatically generated oxygen free radicals is presented. The physiological significance of this process in living cells is suggested.     

Enzyme catalyzed formation of radicals from S-adenosylmethionine and inhibition of enzyme activity by the cleavage products

A large superfamily of enzymes have been identified that make use of radical intermediates derived by reductive cleavage of S-adenosylmethionine. The primary nature of the radical intermediates makes them highly reactive and potent oxidants. They are used to initiate biotransformations by hydrogen atom abstraction, a process that allows a particularly diverse range of substrates to be functionalized, including substrates with relatively inert chemical structures. In the first part of this review, we discuss the evidence supporting the mechanism of radical formation from S-adenosylmethionine. In the second part of the review, we examine the potential of reaction products arising from S-adenosylmethionine to cause product inhibition. The effects of this product inhibition on kinetic studies of ‘radical S-adenosylmethionine’ enzymes are discussed and strategies to overcome these issues are reviewed. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.     

Formation of free radicals during interaction of adriamycin and carminomycin with xanthine oxidase

Xanthine oxidase reduces carminomycin and adriamycin to the semiquinones which have been detected by ESR technique. The steady state carminomycin semiquinone concentration is some tens times higher than the corresponding value for adriamycin. This effect appears to be a result of carminomycin semiquinone stabilization due to internal hydrogen bonding.     

Cell damage by oxygen free radicals

The exposure of isolated and cultured cells to oxygen free radicals generated extracellularly or intracellularly during the metabolism of foreing compounds results in the development of damage that eventually lead to cell death. Multiple mechanisms are involved in these cytopathological processes, including direct attack of free radicals to macromolecules essential for cell life, as well as indirect activation of catabolic processes such as proteases, endonucleases and phospholipases. A key role in triggering these indirect events is played by Ca²⁺ whose cytosolic concentration during oxidative stress raises well above the physiological limits.     

The release of histamine by free radicals

An extensive discussion of the available evidence about the release of histamine by free radicals is presented. In addition, the involvement of the free-radical driven release of histamine in human pathophysiology is debated.