I purchased a commercial kit from Megazyme to assay for catalase activity.
The assay method involves incubating the catalase enzyme sample with a known concentration (65 mM in assay) of hydrogen peroxide. The reaction is stopped by the addition of 15 mM sodium azide. The exact concentration of hydrogen peroxide remaining is measured using an enzyme-linked colourimetric detection method employing 3,5-dichloro-2-hydroxy-benzenesulfonic acid (DHBS), 4-aminoantipyrine (AAP) and peroxidase. The resulting quinoneimine dye is measured at 520 nm.
The expected results should be such that the absorbance values decrease for samples with larger concentrations of catalase. However, I am not able to achieve the expected results as I find it very difficult to obtain consistency in my absorbance values. However, I know that catalase is present in the samples as bubble formation is happening. Additionally, larger number of bubbles are present in reaction wells with higher catalase concentrations.
Would anyone know what I can do to obtain consistency in my results along with the expected decrease in absorbance with increasing catalase concentrations?
Thanks in advance for your help.
Thanks for the tip Andrey.
Yes, I do ensure that there are no bubbles when I add the chromogenic reagent.
Either dilute your enzyme concentration, or add the substrate (or enzyme) when the cuvette is in spectro. Bubbles interfere with the measurements. You have to get rid of them.. In this case dilution seems the only solution because catalase is the one of the fastest enzymes .
Inconsistency in absorbances may also be related with your samples.. Catalase breakdown quickly at room temperature if the protease enzymes did not chelated. (This only raises trouble if you re working with biological tissues)
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