Re: [ccp4bb] Offtopic: FAD enzymatic assay: a little bit more about my enzyme

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

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Subject: Re: Offtopic: FAD enzymatic assay: a little bit more about my enzyme
From: conancao conan_cao {- at -} HOTMAIL {- dot -} COM
Date: 2008-12-02

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Subject: Re: Offtopic: FAD enzymatic assay: a little bit more about my enzyme
From: "Edward A {- dot -} Berry" BerryE {- at -} UPSTATE {- dot -} EDU
Date: 2008-12-02

Subject: Re: Offtopic: FAD enzymatic assay: a little bit more about my enzyme
From: Guenter Fritz guenter {- dot -} fritz {- at -} UNI-KONSTANZ {- dot -} DE
Date: 2008-12-02

Hi Michael,

Fraser et al writes that in case of Synechococcus phytoene desaturase
'NAD+ and NADP+ were observed to be involved, whilst FAD was an
ineffective electron acceptor '
Biochem J. 1993 May 1;291 ( Pt 3):687-92
HTH
Guenter

PS The enzyme itself has no flavin bound?

michael nelson wrote:
> Dear all,
>
> Thank you for all your kind replies.
>
> Here is a little bit more about the enzyme and how I carry out the
> assay at the first place.
>
> My enzyme is a lipid desaturase, originally from plant but
> overexpressed in bacteria. FAD serves as a co-factor for this enzyme,
> in which FAD is reduced to FADH2.
>
> My goal is set up an assay that would allows me to continuously
> monitor the progress of the reaction. And I didn't want to use HPLC to
> analyze the final product since that would take a lot time and we
> don't have an instrument readily available to us. I wish FAD could be
> an alternative way since FAD will have different Abs in reduced or
> oxidized forms.
>
> I set up assay is a regular lab setting (not anaerobic), add FAD,
> substrate, ions and incubate. I finally add enzyme to initialize the
> reaction. I expect to see some decrease of the Ab at 450 nM. But I didn't.
>
> I have several concerns, one is the autooxidisability of FAD, how fast
> FADH2 would be reoxidized by O2 in the air or by the O2 dissolved in
> solution. The second cocern is how fast the FAD reaction will go.
>
> Please advise.
>
> Thank you!
>
> Mike
>
>

--
***********************************

Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz

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

Previous message:
Subject: Re: Offtopic: FAD enzymatic assay: a little bit more about my enzyme
From: conancao conan_cao {- at -} HOTMAIL {- dot -} COM
Date: 2008-12-02

Next message:
Subject: Re: Offtopic: FAD enzymatic assay: a little bit more about my enzyme
From: "Edward A {- dot -} Berry" BerryE {- at -} UPSTATE {- dot -} EDU
Date: 2008-12-02