NADPH oxidase was measured based on its capacity to reduce ferricytochrome
c in ferrocytochrome at pH of 7.8 (22).
The superoxide radical scavenging activity was measured by xanthine and XO using ferricytochrome
c (from equine heart, 3% reduced, Fluka, Switzerland).
COX activity was measured by spectrophotometric method of Hess and Pope  based on the decrease of absorbance on the 550 nm during the oxidation of ferrocytochrome C to ferricytochrome
For spectrophotometric assay of [H.sub.2][O.sub.2], about 500 [micro]l of homogenate was added to the tubes containing 1.5 mM ferricytochrome
. The formation of [H.sub.2][O.sub.2] in the mixture was measured at 550 nm by estimating the oxidation product of ferrocytochrome.
This colorimetric assay is based on an observation of the decrease in absorbance at 550 nm of ferrocytochrome c caused by its oxidation to ferricytochrome
c by cytochrome c oxidase.
Xanthine oxidase (XOD) converts xanthine to uric acid, yielding superoxide anions as a byproduct and subsequently converting ferricytochrome
C directly to ferrocytochrome C, which exhibits absorbance at 550 nm.
Hoppel, "Decreased activities of ubiquinol: ferricytochrome
c oxidoreductase (complex III) and ferrocytochrome c:oxygen oxidoreductase (complex IV) in liver mitochondria from rats with hydroxycobalamin[c-lactam]-induced methylmalonic aciduria," Journal of Biological Chemistry, vol.
UQCRC1 is an oligomeric enzyme that catalyzes the transfer of electrons from coenzyme QH2 to ferricytochrome
c with the coupled translocation of protons across the mitochondrial inner membrane (Brandt and Trumpower, 1994).
In teleost fish, the literature reports methods to determine the reactive oxygen species (ROS) produced during the respiratory burst such as chemiluminescence assay (Verlhac et al., 1996; Verlhac et al., 1998; Cuesta et al., 2002) and reduction of ferricytochrome
C (Jorgensen et al., 1993; Jeney et al., 1997; Santarem et al., 1997; Lee et al., 2004).
Reduction of ferricytochrome
c by superoxide was monitored from the increase in absorbance at 550 nm with reference at 540 nm for 45 minutes .
The reaction mixture contained 50 mM [Na.sub.2]C[O.sub.3]/NaHC[O.sub.3] buffer (pH 10.2), 0.1 mM EDTA, 0.015 mM ferricytochrome
C, and 0.05 mM xanthine.
The use of acetylated ferricytochrome
c for the detection of superoxide radicals produced in biological membranes.