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

- Protein crystallography

Main steps:

- Protein purification
- Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

   - Chemistry
   - Protein
   - Peptide
   - Amino Acids

Xtal community:

- CCP4BB

CCP4bb navigation

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

Previous message:
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

Next message:
Subject: site mutation evaluation
From: Hongmin Zhang hongmin {- at -} RED {- dot -} DFCI {- dot -} HARVARD {- dot -} EDU
Date: 2008-12-02

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

Does the FAD actually dissociate on each turnover, or does it remain bound
and transfer electrons to an acceptor? Succinate dehydrogenase is an
example of the latter, and it can be readily assayed using a mediater
phenazine methosulfate to accept the electrons and transfer them to
a redox dye dichlorophenol-indophenol. The reaction is monitored in
real time by watching the disappearance of the intense blue color of
DCIP, monitored at 600 nm I think. Google for SDH DCIP PMS will probably
give a recipe.

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
>
>

CCP4bb navigation

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

Previous message:
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

Next message:
Subject: site mutation evaluation
From: Hongmin Zhang hongmin {- at -} RED {- dot -} DFCI {- dot -} HARVARD {- dot -} EDU
Date: 2008-12-02