Dehydrogenase assay - (Sep/28/2009 )

Hey, Friends,
I'm setting up the assay for dehydrogenase but have gone through many failures. My enzyme use FAD as reducing agent. But since FADH2 is quickly oxidized to FAD in aerobic condition, I coupled the assay with Phenazine ethosulfate and Dicholorophenolindophenol(DCPIP) for the measurement. However, to my surprise, I found the absorption maximum shifted from 600nm to 650nM after incubation with enzyme. Does anybody has an clue what's happening?

Thanks!

Mike

color shift is what you would expect to see in this type of assay. you could either watch the decrease in absorbance at 600nm or the increase in absorbance at 650nm (i prefer increase).

which dehydrogenase are you using?

Woo, it was so nice to have a very quick reply.

The enzyme I work on is a new dehydrogenase we cloned. I probably can't tell you more at this point. Sorry.

For DCPIP, I thought it was going to be colorless when fully reduced. Do you know where this 650nm absorption come from?

Many thanks!

in your case, color shifts can occur due to environment (eg: pH, ionic strength, specific ions (eg-certain metals), temperature).

Hey, I start doing assays. However, I got another problem. When I simply mix DCPIP and PES with buffer, the absorption at 600nm continue to grow, never really flats out. Could there be anything wrong with my reagent. I'm using phosphate buffer.

dcpip and pes are slowly decomposing. this is natural. you will have to determine your activity from a rising baseline. proper blanking will help.

sometimes you can slow the decomposition by adjusting the conditions (if possible).