Biomedical Engineering Reference
In-Depth Information
Fig. 2. H
2
O
2
emission by mouse brain mitochondria. Incubation medium (37°C) (Subheading 4.2) is supplemented with
4 U/ml horseradish peroxidase, 40 U/ml superoxide dismutase, 0.010 mM Amplex Red, and respiratory substrates as
indicated. (
a
) Mouse brain mitochondria oxidizing succinate. (
b
) Mouse brain mitochondria oxidizing pyruvate and malate.
Additions: Mito, 0.05 mg/ml mouse brain mitochondria; Succinate, 10 mM; Pyruvate:malate, 5:1 mM; Rotenone, 0.5
m
M;
Antimycin, 1
m
g/ml. Numbers near the tracings are the rates of H
2
O
2
production expressed in pmol/min/mg of mitochon-
dria protein.
3.4. Quantifying
the Rates of H
2
O
2
Emission
Calculate the rate of H
2
O
2
emission using the slope coefficient
obtained in step 3.3 by measuring the slope of the fluorescence
traces in relative fluorescence units per minute and dividing it by
the slope coefficient and by the amount of mitochondrial protein
present in the cuvette. Present the rates of H
2
O
2
emission in
pmol/min/mg mitochondria.
3.5. Control
Experiments (Optional)
If desired, two simple control experiments can be performed to
ensure that what is measured is H
2
O
2
and that the reaction is cata-
lyzed by horseradish peroxidase (as opposed to some contami-
nants in your mitochondrial preparations). First, the incubation
buffer can be supplemented with 200-400 U/ml of catalase. This
should greatly suppress the observed rate of H
2
O
2
emission,
although not necessarily completely. Second, 1 mM azide
(a strong inhibitor of horseradish peroxidase) can be included in
the incubation medium; this should suppress the observed rate of
H
2
O
2
emission completely.
Performing the assay as shown on Fig.
2
allows one to estimate the
ROS production from several mitochondrial sites and can be inter-
preted in a reasonably straightforward way as extensively discussed
elsewhere (
17, 24-27, 35, 37, 38
). This protocol yields the rates
of ROS production from Complex I of the respiratory chain in
both forward and reversed electron flux conditions, the rate of
ROS production by the Complex III, and that by the matrix dehy-
drogenases (see (
17
) for discussion). To note, ROS production
3.6. Interpreting
the Obtained Data
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