REMOVEem at NOSPAMunforgettable.com
Wed Apr 30 14:28:57 EST 2003
"Nikolai Scherbak" <nikolai.scherbak at nat.oru.se> wrote in message
news:B8F4773A.7AA%nikolai.scherbak at nat.oru.se...
> Dear colleagues!
> I am a doctoral student and doing kinetic studies on one NADH-dependent
> enzyme that belongs to the SDR-family.
> We are measuring activity using spectrophotometry at 340 nm. Activity
> shows a rapid decrease of absorption in the very beginning (during approx.
> 10 seconds), then decrease dramatically slows but continues during next
> approx 15 min of measurement.
> The slope of this two parts of the curve both depends on concentration of
> the substrate or enzyme.
> Which slope should I use in my calculations of Vmax?
> Thanks for your help,
The first one. You have enough NADH in your reaction, but not enough the
second substrate. Also, you have way too much protein in the reaction.
Delute your protein at least 10 times first, so that you could record the
decrease in absorption as a straight line for at least 1 min. Then, start
increasing your second substrate until you see no more increase in the slope
of the reaction. For determination of kinetic parameters for each of the
substrate, use at least 2 x Vmax concentration of another substrate. 5-30mM
is usually a good starting region.
As for the "second" slope, I think you see it because of some impurities
present in your protein sample - just some other enzyme that uses NADH as a
substrate. There might be also impurities in other components of the
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