About coupled enzyme assay
Park Seung Kyu
skpark at POST.MIYAZAKI-MED.AC.JP
Wed Aug 14 21:18:49 EST 1996
1. I am now doing the enzyme kinetics experiment which adapts coupled
enzyme system. The auxiliary enzyme is pyruvate kinase(PK) and lactate
dehydrogenase(LDH). Once I tried whether the system is proper for my
experiment; that is, I want to confirm the two enzymes are sufficent for
my system. For that, I changed the unit of the PK and LDH added to two
times or three times. I guessed that, first, if the two auxiliary
enzymes are insufficient for my system, the speed of OD decrement(cuase
by NADH oxidation to NAD by LDH) will be faster, and, second, if they
are sufficient there will be no speed changes. But strangely, the the
decrement speed was slower. I cannot explain this phenomenon.
2. I am beginer in kinetics. So, I ask you a basic explanation. When I
read articles or papers about coupled enzyme assay system, I can see
'lag time'. I think it is the time before the system reaches
stedy-state; usually the unit was 'seconds'; and it can be choose by
experimenter by mathmetical calculation using Km or k(velocity
constant). But it is difficult for me to understand. Anyway, I could get
the graphs that shows a linear line flanked by curved line on both ends.
I guess the end time of the initial curve is the lag time; the linear
line means the stedy state; final curve means the end of the reaction
acause by the insufficiency of substrates(I stopped the reaction before
there is no more OD change). So, I think I could get the reaction speed
from the slope of the middle linear line. Is my thinking correct?
Kind explanation would be appreciated..
More information about the Bioforum