Biomedical Engineering Reference
In-Depth Information
10. Small (5-10 ml) glass tubes.
11. Glass (preferable) or plastic disposable cuvettes with all walls
transparent and flat bottom and matching stirring bars.
12. A quartz cuvette.
13. Fluorimeter equipped with a stirred thermostated cuvette
holder such as “Hitachi “F2500, F4700, or F8000 models
(see Note 10).
14. Spectrophotometer capable of quantitative absorbance detec-
tion at 240 nm wavelength.
15. Common labware such as pipettors and tips.
3. Methods
3.1. Prepare the H
2
O
2
Calibration Solution
Fill two glass tubes with 5 ml of de-ionized water, each. To the
first tube, add 5 ml of 30% (v/v) H
2
O
2
solution (1:1,000 dilu-
tion), mix well. Determine the concentration of H
2
O
2
spectro-
photometrically by placing 2 ml of this solution in a quartz cuvette
and reading its absorbance at 240 nm; calculate the concentration
of H
2
O
2
employing the extinction coefficient E
240
= 43.6/M/cm.
Dilute H
2
O
2
solution to ~0.1 mM by taking 50 ml of H
2
O
2
solu-
tion from this tube and adding it to the second tube (1:100 dilu-
tion), mix well. Keep the second tube in ice and use within 1 h to
calibrate the H
2
O
2
assay (see Note 11).
3.2. Calibrate
the Assay
Set the fluorimeter at 555 nm excitation and 581 nm emission
wavelengths and turn on the cuvette holder's thermostat set at
the desired temperature (25-37°C). Fill a cuvette with the incu-
bation buffer (item 2, Subheading
2
), add magnetic stirring bar,
turn on the stirrer, and wait until the cuvette reaches the desired
temperature (25-37°C). Add 4 U/ml of horseradish peroxidase,
10 mM Amplex Red Ultra, 40 U/ml superoxide dismutase
(optional, see Note 12) and mitochondria (0.03-0.1 mg/ml) to
the cuvette. Record the fluorescence changes of Amplex for
~100 s. Make 6-8 additions of ~0.1 mM H
2
O
2
by ~100 nmol
(~1 ml/ml) each, with about 30 s between the additions. You
should obtain a “staircase”-like trace with about equal “steps”
after each addition (see Fig.
1
). Take the fluorescence numbers
approximately at the points indicated by crosses (Fig.
1
). Subtract
the value of fluorescent signal right before the first addition of
H
2
O
2
from all other values and plot them vs. the amount of H
2
O
2
added (Fig.
1
). Calculate the slope coefficient of the graph. This
coefficient is used to calculate the rate of H
2
O
2
production by
mitochondria, in nmols or pmols of H
2
O
2
per mg mitochondrial
protein per minute. The
R
square value should be at least 0.95.
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