NADH determination in enzyme reaction

Peijun ZUO s003206 at mailserv.cuhk.edu.hk
Wed Jun 19 22:11:21 EST 2002


Hi, Everyone

  In my experiment. I will also determine the xylulokinase kinase (XylB)
activity.  Most researchers did the XylB assay according to the method of F.
J. Simpson  (Methods in Enzymology 1966 V9)

 The reaction is procedured as following and the disppearance of NADH was
determined.

D-Xylulose + ATP     D-Xylulokinase       D-xylulose-5-phosphate + ADP

ADP + phosphoenolpyruvate    pyruvate kinase       pyruvate + ATP

 Pyruvate + NADH    Lactate dehydrogenase        actate + NAD+

My question is how did you stop the enzyme reaction. The reaction temp? By
adding what reagent can you inhibite the ATPase and NADH oxidase activities.

 The NADH is not stable. Is that possible to add DTT in reaction mixture to
reduce the oxidation? And in which conc.? I¡¯m afraid by heating at 100 C
for 30s will not inactive the enzymes completely, so I stoped the reaction
by heating at 100 C for 5min. and the result is not good at all.

 Thank you very much for any advice.



Tina S. B

Email address: s003096 at mailserv.cuhk.edu.hk















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