Re: [ccp4bb] Offtopic: FAD enzymatic assay

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Subject: Offtopic: FAD enzymatic assay
From: michael nelson gnomenm {- at -} YAHOO {- dot -} COM
Date: 2008-12-01

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Subject: Re: O/T: can a protein which dimerizes in solution crystallize as a monomer?
From: "R {- dot -} J {- dot -} Lewis" r {- dot -} lewis {- at -} NCL {- dot -} AC {- dot -} UK
Date: 2008-12-02

Subject: Re: Offtopic: FAD enzymatic assay
From: conancao conan_cao {- at -} HOTMAIL {- dot -} COM
Date: 2008-12-01

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: gnomenm@YAHOO.COMSubject: [ccp4bb] Offtopic: FAD enzymatic assayTo: CCP4BB@JISCMAIL.AC.UK

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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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Offtopic: FAD enzymatic assay
From: michael nelson gnomenm {- at -} YAHOO {- dot -} COM
Date: 2008-12-01