I carried out pyrogallol assay to check the activity of SOD1 enzyme. But I got stuck with the calculations. I referred to Marklund's paper in which they mentioned that the amount of enzyme resulted in 50% inhibition corresponding to 1U. From this value how is specific activity calculated? I am not sure about my calculations. I am unable to relate this value with specific activity.
Common activity measurements for SOD are based in indirect assays of superoxide consumption, as pyrogallol auto oxidation or xanthine/XO reduction of CytC. Thus, the calculation of units is always a relative calculation of "SOD units".
The only direct methods that allow estimation of specific activity are pulse-radiolysis or the less common flash photolysis. This protocols use the measurement of superoxide during reaction in the presence of SOD.
Sample SOD calculation:
absorbance of Control at 560 nm= 0.389
The absorbance of Sample at 560 nm = 0.120
Total volume buffer extraction=1mL.
Total reaction volume for read the absorption= 1mL.
Sample tissue weight extraction=0.5 g
% inhibition of NBT reduction by SOD = control OD- treatment OD/ control x 100 =X% inhibition.
50% inhibition is equal to 1 unit of enzyme. then X% is equal to 1/50 x X= Y unit.
SOD calculation will be = (0.389-0.120)/0.389 * 100 =69.15%
50% inhibition =1 Unit of SOD
69.15% inhibition = (1/50)*69.15= 1.38unit
1.38Unit is from =10ul of enzyme extract
Total volume of enzyme extract=1ml=1000ul
(1.38/10 *1000)= 138units of SOD
1ml of extract is from 0.5g of tissue
So SOD units/mg = 138/500= 0.276 units/mg
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