Standards

The arbitrary fluorescence units must be converted to amounts of H2O2. There are different possibilities to do this. One of them is to use H2O2 standards. However, ^M H2O2 solutions are unstable, and they should be prepared just before use from stable mM H2O2 solutions.

An alternative is to use a glucose-glucose oxidase system as standard. In the presence of excess glucose, this couple generates H2O2 at a rate that depends on the amount of glucose oxidase added. For this purpose the following is added to standard tubes: incubation medium, 6 Units/ml of horseradish peroxidase, 0.1 mM homovanillic acid, glucose oxidase, and 14 mM glucose (total volume 1.5 ml). Another advantage of this procedure is that the standards are incubated in parallel in the same conditions as the samples (15 min at 37°C, transfer to the ice-cold bath, and addition of 0.5 ml of 2 M glycine, 2,2 M NaOH, 50 mM EDTA). Glucose oxidase is added in amounts generating 1 nanomol of H2O2/min, and the fluorescence of samples and standards is compared. This is used to calculate the final mitochondrial production of oxygen radicals, which is expressed in nanomoles of H2O2/min. mg of protein. When using this kind of standard, care should be taken that no limiting losses of activity have occurred during transport to or storage of glucose oxidase and horseradish peroxidase at the laboratory.

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