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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 Universitaetsstrasse 10 Postfach M665 D-78457 Konstanz e-mail: [log in to unmask] Tel. Office: +49-(0)7531 88 3205 Tel. Lab : +49-(0)7531 88 3687 Fax: +49-(0)7531 88 2966