Reagents:

1-Tris - HCl - EDTA buffer, (pH 8.0)

 Hydroxy methyl amino methane (0.605 g)

 Ethylene diamine tetra acetic acid (0.0093 g)

Were dissolved in 100 ml dist. H2O.

2. NADPH (0.1 mM)

20 mg dissolved in 5 ml dist. H2O.

3. GSSG (3.3 mM)

0.0075 g dissolved in 0.5 ml dist. H2O

Procedure:

1-The assay mixture was composed of 0.05 ml tris HCl, EDTA buffer, 0.01 ml of homogenate and 0.79 ml H2O

2-Then the mixture was incubated for 10 min at 37°C in water bath.

3- Then 0.1 ml of GSSG was added. The mixture was incubated for another 10 min at 37°C

4-Finally 0.05 ml of NADPH was added. The reaction was inhibited by the addition of NADPH and the decrease of the absorbance at 340 nm due to the formation of NADP+.

5-The change in optical density per minute (Δ A) was read over the linear part of the reaction.

Calculation:

Δ A total volume 1

Enzyme activity = ———X ———————X———————

6.22 sample vol. Wt of tissue used

= unit/g wet tissue.

i want the value in micromole /gram protein