Biomedical Engineering Reference
In-Depth Information
neither particularly reactive (
21
) nor capable of “long-distance”
travel because it does not easily permeate cell and mitochondrial
membranes due to its negative charge (
17, 21
). However, its elec-
trically neutral dismutation product, H
2
O
2
, is more reactive
toward critical cellular targets such as protein SH groups and it is
known to be a precursor of even more aggressive ROS, hydroxyl
radical. Measuring H
2
O
2
emission is a preferred and well-
established method of evaluating mitochondrial ROS production
(
17, 25-35
). It is justified because (see for details (
17
)) most of
the superoxide produced in mitochondria is almost immediately
converted into H
2
O
2
by the matrix-located superoxide dismutase
(MnSOD) or dismutates spontaneously with a high rate into
H
2
O
2
. Moreover, some mitochondrial sites such as flavins may
produce H
2
O
2
directly. Perhaps the most important reason to
measure mitochondrial ROS as H
2
O
2
emission is that methods to
measure it are more quantitative and less labor-consuming and
equipment-demanding than those to detect superoxide.
In this chapter, we describe in details the most recent and reliable
method to measure low levels of H
2
O
2
in vitro. It employs horseradish
peroxidase to trap emitted H
2
O
2
with high selectivity and affinity and
Amplex Red Ultra (a derivative of 10-acetyl-3,7-dihydroxyphenox-
azine) as a sensitive fluorescent probe for H
2
O
2
(
36
).
2. Materials
1. Isolated mitochondria (see Note 1).
2. Incubation buffer: 125 mM KCl, 4 mM KH
2
PO
4
, 14 mM NaCl,
20 mM HEPES-NaOH, pH 7.2, 1 mM MgCl
2
, 0.2% of fatty
acids free bovine serum albumin, and 0.020 mM EGTA (see
Note 2).
3. Amplex Red Ultra (Invitrogen) (see Notes 3, 5).
4. Horseradish peroxidase (e.g. Type VI-A, essentially salt-free,
lyophilized powder, ~1,000 units/mg solid) (Sigma) (see
Notes 4, 5).
5. H
2
O
2
solution in water, 30-32 wt.%, semiconductor grade,
99.999% trace metals basis (Sigma).
6. Respiratory substrates and inhibitors (see Notes 5, 6).
7. (Optional) Catalase (e.g. from bovine liver, lyophilized pow-
der, ~10,000 units/mg protein) (Sigma) (see Notes 5, 7).
8. (Optional) Sodium Azide (Sigma) (see Note 8).
9. (Optional) Cu, Zn Superoxide dismutase (e.g. from bovine
erythrocytes, 2,500-7,000 units/mg protein, lyophilized
powder) (Sigma) (see Note 9).
Search WWH ::
Custom Search