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
