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Enzyme assays

Determination of catalase specific activity

The CAT (EC 1.11.1.6) enzyme activity was measured according to Gallego et al.38 Plant samples were grinded in $\pu{0.1M}\ \ce{KH2PO4}$ buffer (pH $7.0$) and centrifuged at $\pu{4500 rpm}$ for $\pu{5 min}$ (Eppendorf AG bench centrifuge 5417 R, Hamburg, Germany). The supernatant was separated, added with $\pu{73 mM}\ \ce{H2O2}$, and measured at $\pu{240nm}$ through UV/Vis (Perkin Elmer Lambda 14 Spectrometer).

The questions I want to ask are:

  1. Why is $\ce{KH2PO4}$ used?
  2. Why centrifuge it at 4500 rpm?
  3. Why is $\ce{H2O2}$ added to the supernatant?
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As an addition to iad22agp's absolutely correct answer: In catalase assays the decomposition of H2O2 is measured at 240 nm as this is a region where H2O2 absorbs while using rational concentrations. At the same time O2, H2O and CAT (in used concentration) do not absorb. (Primary literature see here)

One unit of catalase will decompose 1.0 µmole of H2O2 per minute at pH 7.0 at 25 °C. The rate of disappearance of H2O2 is followed by observing the rate of decrease in the absorbance at 240 nm. For further calculations see here.

You may also read the helpful literature for this assay which is actually given in your own text. See here: Gallego et al. 1996

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It appears that grinding with phosphate buffer extracts the soluble enzyme catalase, centrifugation separates solid plant material to give a clear solution containing the enzyme to be assayed, and the hydrogen peroxide acts as the substrate for the enzyme. Catalase converts the hydrogen peroxide to oxygen and water. The spectrophotometer is then used to measure the rate of the reaction, and deduce the amount of enzyme present, but what exactly is being detected is not clear.

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