Hi everyone,
I am mesuring catalase from plant leaf with Aebi 1984, and I didn't understand the result.
I am measuring the absorbance at 240 nm and the spectrophotometer records the absorbance every 20 seconds for 2 minutes. I use 10mM 25mM H2O2 phosphate buffer (pH 7.0).
for some samples, the absorbance starts to decrease from the first measurement (so at 00:00 sec) and for other samples it start after 1 minute, what do I need to understand to exploit the results? should I change the protocol?
Thank you for your help
There may be some spontaneous breakdown of the substrate, what happens when you record with substrate but no enzyme?
You might also try using less enzyme so the reaction goes a bit more slowly then you can record for 10 min or so and draw your slope from linear portion and ignore the first and last times points .
There are two points to be considered in general. First, as a spontaneous reaction is expected, commercial kits are much more convenient and sensitive to detect 1micro-U of Catalase activity.
Secondly, if you have a long queue of samples, waiting for their turn, then you will see difference in the initial and final reaction rate in these samples. Consequently, you will see vague measurements.
Thank you very much for your answers Dear Michael J. Benedik I tried to use less enzymes, for some samples it works but not for all, I will try to record for 10 min.
Dear Mehmet KOÇ The protein extract were done one month ago and they were stock at -20°, the mixture was blue when I finished the extraction but now it become grey, I will extract proteine to have fresh extract maybe the first is degraded now.
Dear Saima Rehman I always do my samples one by one, I wait for the first until it finish to prepare the second and try to respect the reaction as described in the article so I don't think that the probleme come from here.
Thank you very much for your answars
- Badges
- Science method