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
Mike:
It seems FAD is readily dissociable for your protein.Then, trust me, you
got to do it anaerobically in order to see the 450nm decrease upon reduction of
the enzyme by the substrate(better use excess substrate).
Sincerely,
Hongnan Cao
UCR
Date: Tue, 2 Dec 2008 11:11:53
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
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
