ON TESTIS AND EPlDlDYMlS OF RATS - Pondicherry University ...

3.21 Determination of antioxidant enzymes in tissues
Activities of antioxidant enzymes such as superoxide dismutase, catalase,
glutathione reductase and glutathione peroxidase were assayed by the methods as
described below.
3.21.1 Assay of superoxide dismutase
Superoxide dismutase (EC 1. IS. 1.1) was assayed by the method of Marklund
and Marklund (1974). The assay mixture contained 2.4 mL of SO mM tris HCI buffer
cont;iininy I mM EDTA (pH 7.6). 300 pL of 0.2 mM pyrogallol (see Appendix 1.20)
and 100 ILL enzyme source. A blank solution was prepared similarly by adding all the
reagents except the enzyme source. Increase in absorbance was measured at 420 nm
against blank at 10 sec intervals for 3 min on Systronics Spectrophotometer.
3.21.2 Assay of catalase
Catalase (EC. 1.11.1.6) was assayed by the method of Claibome (1985). The
assay mixture contained 2.40 mL of phosphate buffer (50 mM, pH 7.0). 10 pL of 19
mM hydrogen peroxide (see Appendix 1.21) and 50 pL enzyme source. Blank was
prepared simultaneously by adding all the reagents except the enzyme extract.
Decrease in absorbance was measured at 240 nm against blank at 10 sec intervals for
3 min on a Systronics Spectrophotometer.
3.21.3 Assay of glutathione reductnse
The activity of glutathione reductase (EC. 1 h.4.2) was assayed by the method
of Carlberg and Mannervik (1975). The assay mixture contained 1.75 mL of
phosphate buffer (100 mM, pH 7.6). 100 pL of 200 mM NADPH, 100 pL of 10 mM
EDTA (see Appendix 1.22). SO pL of 20 mM glutathione oxidized and SO pL enzyme
source. Blank was prepared simultaneously by adding all the reagents except the
enzyme extract. The extinction coefficient of NADPH was measured at 340 nm
against blank at 10 sec intervals for 3 min on a Systronics Specwphotomet~.