Mike:
The trick may be doing the assay under anaerobic condition, especially if
the FAD cofactor is sensitive to oxygen.
You need an anaerobic train and tanometers for the experiment.
Good refs: Hille, R. Biochemistry 1991 Sep 3;30(35):8522-9. Electron
transfer within xanthine oxidase: a solvent kinetic isotope effect study.
Hunt, J., Massey, V. J. Biol. Chem. 1994 Jul
22;269(29):18904-14 Studies of the reductive half-reaction of milk xanthine
dehydrogenase.
Cheers,
Hongnan Cao
UC Riverside
Date: Mon, 1 Dec 2008 18:07:57 -0800From: [EMAIL PROTECTED]: [ccp4bb] Offtopic:
FAD enzymatic assayTo: [email protected]
was working to set up an FAD enzymatic assay. I wished to be able to use 450nM
to continuously monitor the progress of the reaction. The substrate I used is
the natural substrate of the enzyme and the protein is recombinant protein and
I assume it's active since I do see changes in TLC plate. But no signal was
observed at all using a spectrometer. Does anyone here have any suggestions on
how to correctly carry out FAD enzymatic assay? Also I heard from one postdoc
here that FADH2 is quickly reoxidized by O2. Is that right? Should I do the
assay under anaerobic condition? If so, how? Your help is highly
appreciated!Thanks!Best regards,Mike
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