Uv vis double beam spectrophotometer

The enzyme glutathione reductase catalyse the reduction of oxidized glutathione (GSSG) into its reduced form i.e., GSH [54 (link)]. This chemical reaction was simultaneously accompanied by oxidation of NADPH to NADP + . The enzyme activity of tissue sample was assayed by decrease in absorbance of the reaction mixture at 340 nm (UV-VIS double beam spectrophotometer, Systronics, India) as NADPH is progressively converted to NADP + . The enzyme activity was calculated using molar extinction coefficient (6.22 × 103M-1cm-1) and expressed as nmol NADPH oxidized/min/mg protein.

The GPx enzyme was assayed as per the protocol previously suggested with certain modifications [54 (link),55 ]. GPx enzyme catalyses detoxification of hydrogen peroxide and other organic peroxides by oxidising reduced glutathione (GSH). Total GPx activity was measured using cumene-hydro peroxide as substrate whereas selenium-dependent GPx (Se-D GPx) was determined using tert-butylhydroperoxide as substrate at 340 nm by UV-VIS double beam spectrophotometer (Systronics, India). The activity of Se-independent GPx (Se-I GPx) was calculated by subtracting activity of Se-D GPx from that of total GPx. The enzyme activity was also calculated using molar extinction coefficient for NADPH (6.22 × 103M-1cm-1) and expressed as nmol NADPH oxidized/min/mg protein.

The enzyme (GST) was measured by procedure described previously [53 (link)]. GST in tissue sample was quantified by monitoring conjugation reaction of GSH and a broad spectrum substrate of GST called as 1-chloro-2,4-dinitro benzene (CDNB), observing increase in absorbance at 340 nm by UV-VIS double beam spectrophotometer (Systronics, India). The enzyme activity was calculated using molar extinction coefficient for CDNB (9.6 × 103M-1cm-1) and expressed as nmol CDNB conjugated formed/min/mg protein.