Clark type oxygen electrode

Reduction of O₂ by electrons released from NADH by complex I was determined in buffer A* on 5 µl of membranes with a Clark-type oxygen electrode at 30 °C (Hansatech). The reaction was started by adding 1.25 mM NADH while 10 µM piericidin A was used to inhibit the reaction.

Liver and muscle mitochondria were isolated as described (Frezza et al., 2007 (link)). Cytochrome c redistribution and release in response to 40 pmol cBID/mg mitochondria was performed as described (Scorrano et al., 2002 (link)). Mitochondrial oxygen consumption was measured with a Clark type oxygen electrode (Hansatech Instruments) as described (Frezza et al., 2007 (link)). Details on mitochondria Ca²⁺ retention capacity and mitochondrial membrane potential measurements can be found in Supplemental Experimental Procedures.

Direct inhibitors of mitochondrial ETC instantaneously influence mitochondrial oxygen consumption. To assay the effect of EO compounds on oxygen consumption of LtP, a Clark‐type oxygen electrode (Hansatech, Germany) and software MCREC were used. LtP at approximately 10⁸ cells/ml in YEM (25 °C) were added and treated with increasing concentrations of EO compounds between 10 and 200 μM and for EO 5.6–89.6 μg/ml. Each concentration was assayed in quadruplicates, and the results were expressed as percentage of oxygen consumption in comparison with the untreated control LtP. The highest concentration of the vehicle (1% DMSO) caused only 2% inhibition. The uncoupling effect in short‐term and long‐term (0, 6, and 24 hr) incubations with Asc (200 μM) was studied in Oligo‐inhibited (5 μM) and uncoupler‐stimulated (0.5 μM CCCP) LtP in Schneider's medium supplemented with 6 μM hemin. Four replicates were performed for each concentration.

Erv1 enzymatic activity toward oxygen was measured using a Clark-type oxygen electrode (Hansatech Instruments) in 0.5 mL of reaction volume at 25 °C in Tris buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 7.4), as previously described [21 (link)]. When DTT was used as an electron donor, bovine erythrocyte superoxide dismutase 1 (SOD) (Sigma) was added at 10 units/mL to exclude the potential interference of superoxide ions. For Erv1 oxidase kinetic parameter determination, 5 mM DTT was used, so that the DTT concentration was more than 10-fold in excess of the O₂ concentration (~250 µM). The initial slope of the oxygen consumption curve was used to represent the oxygen consumption rate and was calculated by data differentiation using OriginPro software. At least three independent experimental repeats were performed for each experiment. Data were shown as mean ± SEM (n = 3), and differences between groups were statistically analyzed using a Kruskal–Wallis (one-way ANOVA) test.

The mitochondrial oxygen consumption in HL-1 cells was assayed based on the method described by Sun et al., 2018 (link). Mitochondrial respiration buffer (125 mmol/L KCl, 5 mmol/L K2HPO4, 20 mmol/L MOPS, 2.5 mmol/L EGTA, 1 μmol/L Na4P2O7, and 0.1% bovine serum albumin, pH 7.4). Mitochondrial oxygen consumption was measured by Clark type oxygen electrode (Hansatech Instruments, Norfolk, United Kingdom) in mitochondrial respiration buffer at 30°C. Pyruvate (5 mmol/L) and malate (5 mmol/L) were used as substrates for complex I-containing mitochondria at a final concentration of 500 μg protein/ml. ADP-stimulated oxygen consumption (state three respiration) was measured in the presence of 200 μmol/L ADP, and ADP independent oxygen consumption (state four respiration) was also monitored. The respiratory control ratio (RCR, state three divided by state 4) reflects oxygen consumption by phosphorylation (coupling). The ADP/O ratio (number of ADP molecules added for each oxygen atom consumed) is an index of the efficiency of oxidative phosphorylation. State three shows the oxygen consumption of the ADP phosphorylation process during transformation to ATP. State four indicates ADP consumption after the base oxygen consumption and reflects invalid oxygen consumption.

Harvested cells were incubated in the dark for 30 min before measuring their relative O₂ evolution rate of cells under saturated white light illumination using Clark-type oxygen electrode (Hansatech instruments, UK) at 25 °C. The O₂ evolution rate was represented as µmol/mg Chl a/h [22 (link)].

Oxygen evolution was measured using a Clark-type oxygen electrode (Hansatech). Before each experiment, the electrode was calibrated with sterile SOW bubbled with filtered compressed air or N₂ gas for O₂ saturated and O₂ zero standard, respectively. Cells were concentrated 15-fold via gentle filtration onto a 3 μm pore-sized PC filter, resuspended in fresh media, and duplicate subsamples from each biological replicate were introduced to the sample chamber. Samples were briefly bubbled with N₂ before illumination at growth irradiance while O₂ evolution was recorded. Subsequently, samples were exposed to complete darkness, in order to record the rate of respiration. Rates of oxygen evolution and respiration were derived from the slope of the linear increase and decrease of dissolved dioxygen (O₂) over time. The raw data was analyzed using the software Oxypeak supplied by the manufacturer.