Antioxidant and DNA Protection Effects of Taurine by Electron Spin Resonance Spectroscopy - Advances in Experimental Medicine and Biology - page 157

17.2.2 Measurement of Free Radical Scavenging Activity

17.2.2.1 DPPH Radical Scavenging Activity

DPPH radical scavenging activity was measured using the method described by
Nanjo et al. ( 1996 ). Briefly, to a methanol solution of DPPH (to 60 m L, 60 m M) was
added various concentration of taurine sample (60 mL). After mixing vigorously for
10 s, the solution was transferred into a 100 mL Teflon capillary tube, and the scav-
enging activity of each sample for the DPPH radical was measured using an ESR
spectrometer. A spin adduct was measured on an electron spin resonance (ESR)
spectrometer exactly 2 min later. Experimental conditions were as follows: central
field, 3,475 G; modulation frequency, 100 kHz; modulation amplitude, 2 G; micro-
wave power, 5 mW; gain, 6.3 × 10 5 ; and temperature, 298 K.

17.2.2.2 Hydroxyl Radical Scavenging Activity

Hydroxyl radicals were generated by iron-catalyzed Haber-Weiss reaction (Fenton-
driven Haber-Weiss reaction), and the generated hydroxyl radicals rapidly reacted
with nitrone spin-trap DMPO. The resultant DMPO-OH adduct was detectable with
an ESR spectrometer. Briefly, 0.2 mL of taurine sample was mixed with 0.2 mL of
DMPO (0.3 M), 0.2 mL of FeSO 4 (10 mM), and 0.2 mL of H 2 O 2 (10 mM) in a phos-
phate buffer solution (pH 7.2), and then transferred to a 100 m L Te fl on capillary tube.
After 2.5 min, an ESR spectrum was recorded using a JES-FA ESR spectrometer
(JEOL Ltd., Tokyo, Japan). Experimental 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.

17.2.2.3 Superoxide Radical Scavenging Activity

Superoxide radicals were generated by UV irradiation of a riboflavin/ethylenedi-
aminetetraacetic acid solution. The reaction mixtures containing 0.1 mL of
0.8 mM riboflavin, 0.1 mL of 1.6 mM EDTA, 0.1 mL of 800 mM DMPO, and
0.1 mL sample were irradiated for 1 min under a UV lamp at 365 nm. The mix-
tures were transferred to a 100 mL quartz capillary tube and placed in an ESR
spectrometer for measurement. Experimental conditions were as follows: central
field, 3,475 G; modulation frequency, 100 kHz; modulation amplitude, 2 G;
microwave power, 10 mW; gain, 6.3 × 10 5 ; and temperature, 298 K.

17.2.2.4 Alkyl Radical Scavenging Activity

Alkyl radicals were generated by AAPH. The phosphate-buffered saline (pH 7.4)
reaction mixtures containing 10 mM AAPH, 10 mM 4-POBN, and the indicated
concentrations of tested samples were incubated at 37°C in a water bath for 30 min