Evaluation of the horseradish peroxidase-scopoletin method for the measurement of hydrogen peroxide formation in biological systems

163

Mitochondrial swelling and membrane protein thiol oxidation associated with mitochondrial permeability transition induced by Ca 2؉ and inorganic phosphate are inhibited in a dose-dependent manner either by catalase, the thiol-specific antioxidant enzyme (TSA), a protein recently demonstrated to present thiol peroxidase activity, or ebselen, a selenium-containing heterocycle which also possesses thiol peroxidase activity. This inhibition of mitochondrial permeability transition is due to the removal of mitochondrial-generated H 2 O 2 which can easily diffuse to the extramitochondrial space. Whereas ebselen required the presence of reduced glutathione as a reductant to grant its protective effect, TSA was fully reduced by mitochondrial components. Decrease in the oxygen concentration of the reaction medium also inhibits mitochondrial permeabilization andmembraneproteinthioloxidation,inaconcentrationdependent manner. The results presented in this report confirm that mitochondrial permeability transition induced by Ca 2؉ and inorganic phosphate is reactive oxygen species-dependent. The possible importance of TSA as an intracellular antioxidant, avoiding the onset of mitochondrial permeability transition, is discussed in the text.

Section: Fig 2 Effect Of Catalase (F) Tsa (Q) and Ebselen (Oe) Onsupporting

confidence: 61%

“…11 (27). Scopoletin fluorescence was monitored at excitation and emission wavelengths of, respectively, 365 and 450 nm, on a Hitachi F-4010 fluorimeter.…”

Section: Methodsmentioning

confidence: 99%

The Thiol-specific Antioxidant Enzyme Prevents Mitochondrial Permeability Transition

Kowaltowski¹,

Netto²,

Vercesi³

1998

163

“…Determination of Mitochondrial Hydrogen Peroxide GenerationMitochondrial H 2 O 2 production was assessed by the scopoletin oxidation method (13,24).…”

Section: Isolation Of Mitochondria From Rat Bat and Liver-adult Malementioning

confidence: 99%

Identification of a Ca2+-ATPase in Brown Adipose Tissue Mitochondria

Meis

Arruda

Costa

et al. 2006

In brown adipose tissue (BAT) adrenaline promotes a rise of the cytosolic Ca 2؉ concentration from 0.05 up to 0.70 M. It is not known how the rise of Ca 2؉ concentration activates BAT thermogenesis. In this report we compared the effects of Ca 2؉ in BAT and liver mitochondria. Using electron microscopy and immunolabeling we identified a sarco/endoplasmic reticulum (ER) Ca 2؉ -ATPase bound to the inner membrane of BAT mitochondria. A Ca 2؉ -dependent ATPase activity was detected in BAT mitochondria when the respiratory substrates malate and pyruvate were included in the medium. ATP and Ca 2؉ enhanced the amount of heat produced by BAT mitochondria during respiration. The Ca 2؉ concentration needed for half-maximal activation of the ATPase activity and rate of heat production were the same and varied between 0.1 and 0.2 M. Heat production was partially inhibited by the proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and abolished by thapsigargin, a specific ER Ca 2؉ -ATPase inhibitor, and by both rotenone and KCN, two substances that inhibit the electron transfer trough the mitochondrial cytochrome chain. In liver mitochondria Ca 2؉ did not stimulate the ATPase activity nor increase the rate of heat production. Thapsigargin had no effect on liver mitochondria. In conclusion, this is the first report of a Ca 2؉ -ATPase in mitochondria that is BAT-specific and can generate heat in the presence of Ca 2؉ concentrations similar to those noted in the cell during adrenergic stimulation. BAT3 is capable of rapidly converting fat stores to heat and has been used as a model system for the understanding of nonshivering heat production and mechanisms of energy wasting to control obesity (1-9). The signal that activates the rate of heat production in BAT cells is the rise of the cytosolic Ca 2ϩ concentration from a basal level of 0.05 M up to the range of 0.2-0.7 M. This is promoted by ␣ 1 -and ␤ 3 -adrenergic receptors located in the cell membrane. Activation of ␣ 1 -adrenoreceptors leads to the release of Ca 2ϩ from intracellular stores into the cytosol, whereas ␤ 3 -adrenergic receptors promote the release of free fatty acids and increase the effect of Ca 2ϩ release induced by ␣ 1 -adrenoreceptors (10, 11). At present we do not know how the rise of the cytosolic Ca 2ϩ concentration activates the rate of heat production in BAT cells.In a previous report (12) we identified a sarco/ER Ca 2ϩ ATPase (SERCA 1) in vesicles derived from BAT ER. In this report we show that the rate of heat produced by BAT mitochondria is enhanced when the Ca 2ϩ concentration in the medium is raised to a level similar to that observed in BAT cells during adrenergic stimulation. This effect is not observed in liver mitochondria, a tissue that is not specialized in heat production. EXPERIMENTAL PROCEDURES Isolation of Mitochondria from Rat BAT and Liver-Adult maleWistar rats were killed by decapitation. Mitochondria were isolated as previously described (13). Briefly, interscapular BAT and liver were removed and homogenized in a mix...

“…Glucose oxidase was placed in dialysis bags (molecular weight cutoff of 3,500), which were added just prior to experiments to different flasks containing either 0.2 mM oxyhemoglobin or 5% (v/v) intact RBC in 5 mM glucose and Krebs-Ringer phosphate buffer (pH 7.4) at 37°C. The rate of H 2 O 2 production was measured spectrophotometrically at 402-417 nm in a Shimadzu UV-3000 double beam, dual wavelength spectrophotometer using the horseradish peroxidase assay according to Boveris et al (25). The presence of the dialysis bag did not significantly affect the rate of hydrogen peroxide production by the glucose-glucose oxidase system.…”

Section: Incubation Conditionsmentioning

confidence: 99%

Mechanism of the Formation and Proteolytic Release of H2O2-induced Dityrosine and Tyrosine Oxidation Products in Hemoglobin and Red Blood Cells

Giulivi

Davies

2001

111

Oxyhemoglobin exposed to a continuous flux of H 2 O 2 underwent oxidative modifications, including limited release of fluorescent fragmentation products. The main fragments formed were identified as oxidation products of tyrosine, including dopamine, dopamine quinone, and dihydroxyindol. Further release of these oxidation products plus dityrosine was only seen after proteolytic degradation of the oxidatively modified hemoprotein. A possible mechanism is proposed to explain the formation of these oxidation products that includes cyclization, decarboxylation, and further oxidation of the intermediates. Release of dityrosine is proposed as a useful technique for evaluating selective proteolysis after an oxidative stress, because dityrosine is metabolically stable, and it is only released after enzymatic hydrolysis of the oxidatively modified protein. The measurement can be accomplished by high performance liquid chromatography with fluorescence detection or by high efficiency thin layer chromatography. Comparable results, in terms of dityrosine release, were obtained using red blood cells of different sources after exposing them to a flux of H 2 O 2 . Furthermore, dityrosine has been reported to occur in a wide variety of oxidatively modified proteins. These observations suggest that dityrosine formation and release can be used as a highly specific marker for protein oxidation and selective proteolysis.