I am estimating activity of CAT using spectrophotometer. The reaction mixture (1 ml) contains 850 uL of 50 mM phosphate buffer (pH 7.2), 100 uL of 200 mM H2O2 and 50 uL of enzyme extract. The absorbance is read at 240 nm at an interval of 5 seconds up to 120 seconds. Instead of linear decrease in OD, I am getting uneven pattern of absorbance. The OD decreases for 10-15 seconds, then increases and later follows zigzag pattern.
Upon doubt, I run a blank by adding only phosphate buffer and H2O2. I was expecting that I would get constant values or might observe slight decrease in absorbance. However, this time too, I observed zigzag pattern of OD.
I don’t understand why I am getting such pattern even when I am not adding the enzyme extract?
Solutions I tried:
(1) Use of quartz cuvette, not glass cuvette
(2) I replaced 200 mM H2O2 (prepared from commercially available 30% H2O2) with 3% H2O2 (sigma), but again same result.
(3) I don’t have doubt on sensitivity of spectrophotometer as it give correct readings during other assays
Hello,
at 240 nm you MUST use quartz cuvette. H2O2 concentration is unlikely the problem in you case. I suspect spectrophotometer sensitivity and enzyme interference. The apparatus may work well in some wavelength but not in others. Try following for this case:
set 240 nm wavelength. Also, set the apparatus to continuous reading. Check if the absorbance is constant, without quartz cuvette, with it, with distilled water and with phosphate buffer. If the apparatus is OK, the absorbance should be constant or with minor changes in all tests.
To find if there is any interfering agent is enzyme extract, you should check if the test works well with standard pure catalase enzyme or not. If not, you are doing the test wrong or CAT standard is not OK.
It is obvious that the problem makes bubbles of oxygen generated in reaction hydrogen peroxide and catalase. Try to decrease the concentration of H2O2 or the concentration of the sample.
If there is no result, maybe the attached procedure could help to solve the problem.
I agree with Vesna that the formation of bubbles is problematic. You should use either a quartz cuvette, as Seyyed suggested, or UV-transparent plastic cuvettes which are disposable (so no cleaning required) and much cheaper. You can purchase a pack of 100 for less than 90 $ from some vendors.
Cheers,
Christian
Here is description of method. I hope it help you rid off problems.
http://www.ncbi.nlm.nih.gov/pubmed/11185567
Hello,
First of all make sure that the UV light is on in your spectrophotometer. Though I don't know how your spectrophotometer works, in some you need to manually switch the lamp even after setting the wavelength required. After switching on, leave at least half an hour to warm up. Usually, assays involving UV can be bit inconsistent few times.
And coming to H2O2 concentration, I use lesser than yours. But that shouldn't be a problem , it should decrease evenly no matter what concentration you use i believe.
If you observe the bubbles in your cuvette after reading, then the enzyme is definitely working. It's the problem with other stuffs, mainly the instrument.
Hope it helps. Good luck.
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