Plant enzyme stablility and assays

Simon Hopkins S.A.Hopkins at biosci.hull.ac.uk
Mon Nov 18 06:31:40 EST 1996


As part of my PhD I'm currently assay enzymes from various plant 
seedlings. One of the enzymes is causing me problems since both the 
literature and experiment suggest that the enzyme is loses its activity 
between -20c and room temp. (overnight at 20c, and 2hrs at room temp). 
This even occurs in frozen plant tissue. Can anyone suggest why this 
should occur even at low temps and any ways to make the enzyme more 
comfortable in its surroundings.

Also the assay relies on a UV absorbance change of NADH to NAD+ via a 
coupled enzyme system (with malate dehydrogenase). A partially-purified 
enzyme solution (instability prevents complete purification) contains 
endogenous NADH oxidase which gives rather a high background rate 
whithout the addition of enzyme substrates. Whilst it is not essential 
I'd like to inhibit the endogenous NADH oxidase (it would make me sleep 
better!). Any ideas???

Thanks for all your help,

Geordie Si.





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