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
--
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