About coupled enzyme assay

Park Seung Kyu skpark at POST.MIYAZAKI-MED.AC.JP
Wed Aug 14 21:18:49 EST 1996


Dear Everyone:

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..

Sincerely yours,

SK Park



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