Inactivating enzymes before [NADH] assay

engelbert_buxbaum4 at my-deja.com engelbert_buxbaum4 at my-deja.com
Wed Nov 8 01:24:25 EST 2000


In article <3A0887DD.55A67663 at bimcore.emory.edu>,
  Tim Barnett <tbarnett at bimcore.emory.edu> wrote:
> Hi all,
>
> I am interested in performing a coupled NAD-dependent enzyme assay
where
> I will measure the amount of NADH produced after a period of time.
> However, I need a way to inactivate the enzyme without affecting the
> NADH in the reaction mixture. It appears that NADH is sensitive to
both
> acid and heat, which are the only strategies I can think of. If anyone
> has any ideas on how to do this I would be very thankful.


I am not quite sure what you want to do here. If it is just measuring
the time dependency of NAD -> NADH conversion, thats easy: These
compounds have different absorbance at 340 nm, so you can simply use a
spectrophotometer and a recorder to perform this measurement, without
inhibition at all. If you have no recorder, use a stopwatch and read
every, say, 30 sec.

NAD and NADH have different sensitivities to pH, one is sensitive to
alkali, the other to acid (don't remember which is which, you'll have to
look that up). This allows the selective destruction of either one of
them.

A good way to inhibit enzyme activity is the addition of denaturing
agents (in particular SDS, but also urea, guanidinium chloride etc).
Brief insertion into a boiling water bath might also work, you'll have
to check whether your cofactors survive this.


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