Hello every body,
I would like to measure level of Catalase activity in PC12 cells after lead exposure. I used phosphate buffer and H2O2 method to measure that. I have not get any deception in H2O2 after homogenize the cells and centrifuge them for 20min.
Please any help or suggestion to make my enzyme available to the substrate
Dear Sarah,
I have read the attached paper and found that it to be helpful in answering your question.
Please read the experimental method. I have copied the paper's abstract for your quick look.
Abstract: The antioxidative properties of a novel curcumin analogue
(2E,6E)-2,6-bis(3,5-dimethoxybenzylidene)cyclohexanone (MCH) were assessed by several in vitro models, including superoxide anion, hydroxyl radical and
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and PC12 cell protection from H2O2 damage. MCH displayed superior O2 quenching abilities compared to curcumin and vitamin C. In vitro stability of MCH was also improved compared with curcumin. Exposure of PC12 cells to 150 µM H2O2 caused a decrease of antioxidant enzyme activities, glutathione (GSH) loss, an increase in malondialdehyde (MDA) level, and leakage of lactate dehydrogenase (LDH), cell apoptosis and reduction in cell viability. Pretreatment of the cells with MCH at 0.63–5.00 µM before H2O2 exposure significantly attenuated those changes in a dose-dependent manner. MCH enhanced cellular expression of transcription factor NF-E2-related factor 2 (Nrf2) at the transcriptional level. Moreover, MCH could mitigate intracellular accumulation of reactive oxygen species (ROS), the loss of mitochondrial membrane potential (MMP), and the increase of cleaved caspase-3
activity induced by H2O2. These results show that MCH protects PC12 cells from H2O2 injury by modulating endogenous antioxidant enzymes, scavenging ROS, activating the Nrf2 cytoprotective pathway and prevention of apoptosis.
Hoping this will be helpful,
Rafik
Thank you so much Rafik,
Have you done CAT activity measurement before? how much reduction in absorbance I should expected?
Dear Sara,
I have done this kind of measurements many years ago when I was young such as you.
I suggest that you run positive control with Vitamin C (EC50 about 100 μM). I expect the reduction to be more than 50% (it is just guess).
Good luck,
Rafik
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