Biology Reference
In-Depth Information
and then transferred to a 100 mL quartz capillary tube. The spin adduct was recorded
on an ESR spectrometer. Measurement conditions were as follows: central field,
3,475 G; modulation frequency, 100 kHz; modulation amplitude, 2 G; microwave
power, 1 mW; gain, 6.3 × 10 5 ; and temperature, 298 K.
17.2.3
DNA Nicking Assay
The method of hydroxyl radical-induced DNA breakage in plasmid pBR322 was
modified from Kitts and others (2000). Briefly, 2 mL of taurine (1 mg/mL) was
mixed with 2 mL of EDTA-Na 2 (10.09 g/L), KH 2 PO 4 buffer (0.05 mol/L, pH 7.4),
H 2 O 2 (1.02 g/L), FeSO 4 (2.42 g/L), and 0.1 mg/mL of pBR322 plasmid DNA in a
500 mL micro-centrifuge tube. The molar ratio of FeSO 4 /EDTA was kept at 0.53.
The final volume of the reaction mixture was brought to 12 mL with deionized
distilled water and incubated for 1 h at 37°C. Following incubation, 3 m L of
autoclaved distilled water and 3 mL of 6 × DNA loading dye (Fermentas) was
loaded onto a 1.2 g/100 mL of agarose gel. Electrophoresis was conducted at
80 V in a Tris-acetate-EDTA.Na 2 (TAE) buffer (7.25 g/L of Tris-acetate and
0.29 g/L of EDTA, pH 7.4) using a horizontal submarine gel electrophoresis
apparatus (Atto Corp., Yushima, Bunkyo, Tokyo, Japan). After electrophoresis,
the gel was stained with ethidium bromide (0.5 mg/mL in deionized distilled
water) for 10-15 min with gentle shaking followed by destaining with distilled
water until the background was clear. DNA bands were visualized under illumi-
nation of UV light and photographed with Gel Doc system (Ultra-Violet Products
[UVP] Ltd., Cambridge, UK).
17.2.4
Statistical Analyses
All data were expressed as the mean ± SEM, and statistical analyses were performed
using Statistical Analysis System version 8.0 (SAS Institute, Cary, NC, USA). The
differences between means were assessed by the Student's t -test, and statistical
signi fi cance was de fi ned at P < 0.05.
17.3
Results
17.3.1
Free Radical Scavenging Activity
In the present study, the radical scavenging activities of taurine at various concentrations
were investigated using DPPH, hydroxyl, alkyl, and superoxide radical scavenging
properties measured using an ESR spectrometer. DPPH is a stable radical that is used to
Search WWH ::




Custom Search