concerning activity of the enzymes

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mirka
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concerning activity of the enzymes

Post by mirka » Thu Nov 08, 2007 12:29 pm

I've started new assay with squalene epoxidase which converts radioactive substrate (squalene) into radioactive product (oxidosqualene). Let say that I need to this reaction 50 000 dpm which is in 1nM of squalene but the concentration of the squalene which is needed to Vmax is 8uM. What do you think, should I add the appropraite amount of "cold" squalene to this radiactive one or not?

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canalon
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Post by canalon » Fri Nov 09, 2007 2:43 am

As long as you know the ratio cold/hot in the starting product, you can convert thhe amount of produce back, assuming that the cold and hot squalene have the same properties and are thus degraded at similar rate.
Patrick

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blcr11
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Post by blcr11 » Fri Nov 09, 2007 12:57 pm

I think it depends on how you're using the radioactive substrate. If you are using it as a tracer, then you add cold substrate to the level of activity you want to support (8 uM if you like) and add the tracer to the amount of dpm's you need. The contribution of tracer to the total concentration of substrate is negligible. You would use the radioactivity as a monitor of %conversion which will range from 0-100%. But if you are going to isolate the product and look at the distribution of isotope by, say, mass spectroscopy, or nmr, or something like that, you probably don't want to dilute out the isotope so much. I don't know how you intend to do you analysis.

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