Dehydration of H2CO3 by carbonic anhydrase - please help!

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Dehydration of H2CO3 by carbonic anhydrase - please help!

Post by voleman » Thu Nov 16, 2006 10:13 pm

Hello everyone,

I'm a 1-year A-level student and I'm sorting out my AS coursework at the moment. I'd like to measure the effect of temperature on carbonic anhydrase activity but my college tells me that the pure enzyme is too expensive to procure. I need therefore to find some body tissue which is packed full of it and I've managed to establish that lung tissue (understandably) contains a lot of enzyme, as do erythrocytes but I'm not sure how I should prepare the tissue.
I intend to homogenise the tissue but I'm also thinking of using a lysing agent. I don't really know anything about them though. My questions really are:

- Should I use spleen or lungs (assuming I can get hold of both)?
- Is lysis of the cells actually necessary?
- If so, what is the best kind of lysing agent to use? pH neutral or slightly acidic?
- How can I be sure that the lysing agent won't denature the enzyme?
- Lastly, has anyone done anything similar before? I can find very little information on this particular experiment and would appreciate input from someone who has used this enzyme before.

I forgot to say: my results will be obtained by measuring the amount of CO2 evolved.



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Post by weesper » Sun Nov 19, 2006 11:28 pm

I would take a kidney tubular epithelium cell line (eg MDCK) and then use that measure pH changes. Not sure if would work though (no lysis necessary just measure it in the cultute medium).

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