Catalase activity was measured in a reaction mixture (1 ml) containing 100 mM Na2HPO4 buffer pH 7.4, 10mM H2O2, 20ug protein extract. I took reading at od240 every 5 sec. got readings 0.47, 0.402, 0.342, 0.29, 0.241, 0.197, 0.161, 0.128, 0.1, 0.073, 0.053. Specific activity of catalase was calculated from the equation: specific activity (units/mg of protein/min) = ΔA 240 nm (1 min) × 1000/43.6 × mg protein. and got the value of 935.7798165. Would appreciate if any one can tell me if I have made any mistake in calculations or procedure. Or
Can any one explain me the correct way to calculate specific activity of catalase....
Hi there,
Specific activity is calculated using initial velocity data. Therefore first you should trace the kinetics OD=f(t). By doing so you see that the signal is not linear towards the time of the full experiment. So I only considered the five first timepoints for a period of time of 20s where the variation is linear. The decrease observed is (0.47-0.241)=0.229 in 20s so it would be 0.687 per minute. If I introduce this data in your formula and using 0.02mg of protein (0.687*1000/(43.6*0.02)), it gives: 787.84 U/min/mg... Which is a bit different from your calculation.
Hi there,
Specific activity is calculated using initial velocity data. Therefore first you should trace the kinetics OD=f(t). By doing so you see that the signal is not linear towards the time of the full experiment. So I only considered the five first timepoints for a period of time of 20s where the variation is linear. The decrease observed is (0.47-0.241)=0.229 in 20s so it would be 0.687 per minute. If I introduce this data in your formula and using 0.02mg of protein (0.687*1000/(43.6*0.02)), it gives: 787.84 U/min/mg... Which is a bit different from your calculation.
Units are usually expressed as micromoles of prduct formed per min per mg of protein. Does you calculation convert Delta A240 to micromoles using the factor 1000/43.6? Does this conversion take into account the path length of the cuvette and the extinction coefficient change?
Thank you Dominique Liger and Adam B Shapiro for your quick reply... I have recorded the time required for the A240 to decrease from 0.45 to 0.40 absorbance units every 5 secs, as suggested in the paper Lack et. al. 1965. So I took only first 2 values as it was within the range 0.45 to 0.40.
Yes I think I did convert Delta A240 to micromoles using the factor 1000/43.6.
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