The extraction was made according to Aebi 1984. A complete reaction mixture contains 1.5 ml of 100 mM potassium phosphate buffer (pH 7.0), 0.5 ml of 75 mM H2O2, 0.2 ml of enzyme extract
and 0.8 ml of distilled water was used.
In case of reading on Spectro at 240nm no linear decrease was observed despite continues up down was noted in the values. What could be the possible reason?. How we can come up with??
If you are not getting enzyme activity, either the enzyme is dead or your reaction conditions are wrong. The best way to differentiate is to use a positive control- enzyme which is known to be working. Add it to your system in place of the sample and see if you get the expected decrease in absorbance.
The more common reason:
1. Lack of substrate ( H2O2)
2. Incorrect enzyme concentration
3. Incorrect wavelength
4. Inactive enzyme
5. Substrate depletion before measurement
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