Svetlana Trivić

The chemical mechanism of action of glucose oxidase from Aspergillus niger

Glucose oxidase from Aspergillus niger (EC 1.1.3.4) is able to catalyze the oxidation of β-D-glucose with p-benzoquinone, methyl-1,4-benzoquinone, 1,2-naphthoquinone, 1,2-naphthoquinone-4-sulfonic acid, potassium ferricyanide, phenazine methosulfate, and 2,6-dichloroindophenol. In this work, the steady-state kinetic parameters, V1/KB, for reactions of these substrates were collected from pH 2.5–8. Further, the molecular models of the enzymes active site were constructed for the free enzyme in the oxidized state, the complex of β-D-glucose with the oxidized enzyme, the complex of reduced enzyme with methyl-1,4-benzoquinone, the reduced enzyme plus 1,2-naphthoquinone-4-sulfonic acid, oxidized enzyme plus reduced 1,2-naphthoquinone-4-sulfonic acid (hydroquinone anion), and oxidized enzyme plus fully reduced 1,2-naphthoquinone-4-sulfonic acid.Combining the steady-state kinetic and structural data, it was concluded that Glu412 bound to His559, in the active site of enzyme, modulates powerfully its catalytic activity by affecting all the rate constants in the reductive and the oxidative half-reaction of the catalytic cycle. His516 is the catalytic base in the oxidative and the reductive part of the catalytic cycle. It was estimated that the pKa of Glu412 (bound to His559) in the free reduced enzyme is 3.4, and the pKa of His516 in the free reduced enzyme is 6.9.

Antioxidant Capacity of Ocimum basilicum L. and Origanum vulgare L. Extracts

The antioxidant properties of five different extracts (Et2O, CHCl3, EtOAc, n-BuOH, and H2O) of Ocimum basilicum L. and Origanum vulgare L. were studied. Antioxidant activity was assessed in six different model systems. Free radical scavenging capacity (RSC) was evaluated by measuring the scavenging capacity of extracts on DPPH, NO, O2•− and OH radical, as well as on hydrogen peroxide (H2O2). In addition, the protective effects on lipid peroxidation in liposomes (LPx) were evaluated by TBA-assay using the Fe2+/ascorbate induction system. The amount of total phenolic compounds and content of total flavonoids was also determined. EtOAc, n-BuOH and H2O extracts of O. basilicum and O. vulgare expressed very strong scavenger activity. Furthermore, the mentioned extracts showed notable inhibition of LPx. On the other hand, Et2O and CHCl3 extracts showed much weaker effect in the neutralization of DPPH, NO and O2•− radicals and the neutralization of H2O2. When examining the production of OH radicals and inhibition of LPx, the Et2O and CHCl3 extracts showed weak prooxidative properties. The observed differences in antioxidant activity could be partially explained by the levels of phenolics and flavonoids in the investigated O. basilicum and O. vulgare extracts.

Antioxidant Activities of Celery and Parsley Juices in Rats Treated with Doxorubicin

We have examined the influence of diluted pure celery and parsley leaf and root juices and their combinations with doxorubicin on the antioxidant status [as measured by the content of reduced glutathione (GSH) and ferric reducing antioxidant power (FRAP)] in liver homogenate and hemolysate and on the contents of cytochrome P450 in liver homogenate. It was found that doxorubicin significantly decreased the content of reduced glutathione and the total antioxidative status (FRAP) in liver homogenate and hemolysate, while celery and parsley juices alone and in combination with doxorubicin had different actions. Doxorubicin and celery juice had no effect on content of cytochrome P450. However, in combination with doxorubicin, parsley root juice significant increased, and parsley leaves juice decreased the cytochrome P450 content (compared to doxorubicin treated animals). Only parsley root juice significantly increased the content of cytochrome P450.

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 pKa values have been ascribed to protonation forms of most of the above mentioned complexes of yeast enzyme with coenzymes and substrates.

Reduction of aryl-nitroso compounds by pyridine and flavin coenzymes

1. A systematic kinetic investigation of the reduction of aryl-nitroso compounds by pyridine and flavin coenzymes and their analogs, in enzymatic and nonenzymatic systems, has been reported. 2. Two main groups of nitroso compounds have been investigated, representatives nitroso-benzene and 1-nitroso-2-naphthol; in all enzymatic and nonenzymatic systems, the former was always reduced to phenyl-hydroxyl-amine and the latter to 1-amino-2-naphthol. 3. Pyridine compounds included NADH, APAD-4H2 and DBNA-4H2 in nonenzymatic systems, and liver alcohol dehydrogenase. Flavin compounds included 1,5-dihydrolumiflavin and various forms of reduced 5-ethyl-lumiflavin, in nonenzymatic systems, and the flavoenzymes glucose-oxidase and NADPH-cytochrome P450 reductase. 5. Pyridine coenzymes and their analogs reduced nitroso compounds by a direct hydride transfer, with a primary kinetic isotope of 9.5 +/- 2.2. 6. All flavin compounds (glucose-oxidase and its nonenzymatic analog 1,5-dihydrolumiflavin and NADPH-cytochrome P450 reductase and its analog 5-ethyl-1,5-dihydrolumiflavin) reduced aryl-nitroso compounds with high efficiency (k2 greater than 10(5)M(-1) min(-1)). 7. The flavin compounds have been shown to be much more efficient reductans of nitroso compounds, compared to pyridine coenzymes, both in enzymatic and nonenzymatic systems; the only exception to this rule presented the extremely efficient reduction of p-substituted aryl-nitroso compounds by liver alcohol dehydrogenase.

Antioxidant effects of some drugs on immobilization stress combined with cold restraint stress.

The aim of this work was to investigate the effect on antioxidant potential of some commonly used drugs (morphine, tramadol, bromocriptine, haloperidol and azithromycin) on immobilization stress (IS) combined with cold restraint stress (CRS) in the rat. After the drug treatment the animals were kept immobilized in the cold chamber at 4±0.3ºC for 3 hours and then decapitaed and the livers were extracted. The following parameters were determined in the liver homogenate: content of reduced glutathione, activities of catalase, xanthine oxidase, glutathione reductase, glutathione peroxidase, peroxidase, and lipid peroxidation intensity. A battery of biochemical assays was used and the resulting data were statistically analyzed. Combined stress exhibited a prooxidative action (increased catalase activity, lowered content of reduced glutathione). Significantly enhanced catalase activity that was observed in all groups compared to the control indicates that the primary reactive oxygen species (ROS) metabolite is hydrogen peroxide, which decomposes very rapidly (very high catalase activity), thus hindering formation of OH radicals as the most toxic ROS. None of the tested drugs showed a protective effect on combined IS and CRS. The intensity of lipid peroxidation did not change either in the combined stress or under additional influence of the drugs. Probably, under our experimental conditions, the time was not sufficiently long to observe damage of lipid membrane by ROS.

Effects of Various Drugs on Alcohol-induced Oxidative Stress in the Liver

The major aim of this work was to investigate how alcohol-induced oxidative stress in combined chemotherapy changes the metabolic function of the liver in experimental animals. This research was conducted to establish how bromocriptine, haloperidol and azithromycin, applied to the experimental model, affected the antioxidative status of the liver. The following parameters were determined: reduced glutathione, activities of glutathione peroxidase, glutathione reductase, peroxidase, catalase, xanthine oxidase and lipid peroxidation intensity. Alanine transaminase was measured in serum. Alcohol stress (AO group) reduced glutathione and the activity of xanthine oxidase and glutathione peroxidase, but increased catalase and alanine transaminase activity. The best protective effect was achieved with the bromocriptine (AB1 group), while other groups had similar effects on the studied parameters.

Anthracycline-based combined chemotherapy in the mouse model

SummaryOur research was aimed at establishing if and how selenium (Se) ion, N-acetylcysteine (NAC), sodium salt of monoketocholic acid (MKH) and superoxide-dismutase (SOD), administered in the experimental animal model, could affect the possible cytotoxicity as-sociated with anthracycline-based combined chemotherapy with doxorubicin, vincristine and prednisolone (DVP). The following biochemical parameters were investigated: the extent of lipid peroxidation (LPx), and the activity of peroxidase (Px), catalase (CAT), glutathione-peroxidase (GSHPx), and xanthine-oxidase (XOD). A statistical increase in LPx activity was obtained by SOD, MKH, DVPSe and DVPMKH. All chemotherapeutic agents reduced Px activity in a statistically significant manner. There was no statistical significance for the results regarding the effects of the administered substances on GSHPx activity. The results for DVP, SOD, MKH, DVPSOD, DVPSe and DVPMKH showed reduced XOD activity which was statistically significant, which was lowest in the case of MKH, while NAC and Se reduced the activity of this enzyme but statistically non significant. NAC, Se, DVP, MKH and DVPMKH caused a reduction in CAT activity, while DVPSOD and DVPSe caused an increase of the latter.

Effects of St John's Wort (Hypericum perforatum L.) Extracts on Epileptogenesis

The purpose of this study was to investigate the effects of treatment with water, n-butanol and ether extracts of Hypercom perforatum L. on epileptogenesis in rabbits. Animals from the control group received solvent-ethanol, and the kindling model of epilepsy was used. Epileptic focus was induced in Chinchilla rabbits by stimulation of the hippocampus. The following parameters were determined: the minimum current strength necessary to induce after-discharge (AD) – discharges appearing after cessation of stimulation; AD duration; the number of stimulations necessary to induce spontaneous kindling; and the latency time for the development of full kindling. The results obtained indicate that epileptogenesis is influenced by Hypericum perforatum L. extract treatment.Animals treated with an ether extract of Hypericum perforatum L. required significantly weaker minimum current strengths for the development of epileptogenic focus, and displayed longer AD times, while the number of electro-stimulations necessary for full kindling was less. In contrast, animals treated with water and n-butanol extracts required increased electro-stimulations for the development of epileptic discharge, and displayed shortened AD durations versus controls.

Antioxidant Effects of Some Drugs on Ethanol-induced Ulcers

The aim of this work was to investigate the antioxidant potential of some commonly used drugs (bromocriptine, haloperidol and azithromycin) on alcohol-induced ulcers in the rat. The following parameters were determined: content of reduced glutathione, activities of catalase, xanthine oxidase, glutathione reductase, glutathione peroxidase, peroxidase, and lipid peroxidation intensity. A battery of biochemical assays were used and the resulting data was statistically analyzed. Alcohol stress caused gastric ulcerations and hemorrhages and changed all the examined parameters except glutathione peroxidase activity. All drugs reduced the ulcer index and hemorrhages, with azithromycin showing the strongest effects. The drugs in combination with alcohol showed different effects on biochemical parameters. Our results indicate that the gastroprotective effects of the investigated drugs on experimental lesions induced by 100% ethanol could not be correlated with their antioxidative properties.