From: Bruce Allan Palfey (brupalf$##$umich.edu)
Date: Tue Dec 19 2000 - 10:09:28 EST
Dear Joanne
Because reduced flavins react readily with oxygen, it is very important to
be rigorously anaerobic. Under anaerobic conditions, a number of reducing
systems have been used, such as titrating with dithionite or titanium
chloride, or photoreduction using carboxylates such as EDTA or glycine as
electron donors in the presence of catalytic deazaflavin. NAD(P)H is very
slow in the absence of an enzyme. There are a few enzymes reported whose
purpose appears to be to reduce flavins in solution by NAD(P)H (flavin
reductases), but actually many flavoenzymes will catalyze the reduction of
free flavins (at a low rate) in the absence of alternate oxidizing
substrates. Borohydride sometimes has the problem (at least with enzymatic
systems) of unnatural flavin reduction products.
As for checking the "activity" of FAD, it depends on what interests you.
FAD could break down to/be contaminated with FMN and riboflavin.
However, all that is needed for flavin redox chemistry to occur in
solution is the isoalloxazine moiety, and there will likely be no
difference in "activity" if your FAD is contaminated with FMN and
riboflavin. Of course, the difference is critical when enzymatic systems
are considered, but I get the impression you're not dealing with enzymes.
HPLC methods for analyzing flavins have been reported: Anal. Biochem.
(1980) 109, 87-93; Methods in Enzymology (1986) 122, 199-209; Flavoprotein
Protocols, Chapman & Reid, eds., Humana Press, 1999. TLC on silca gel
using butanol:acetic acid:water (12:3:5) also works.
ciao,
Bruce
Bruce Palfey
Department of Biological Chemistry
University of Michigan
Ann Arbor, MI, 48109-0606
On Tue, 19 Dec 2000, Joanne Jamie wrote:
> Dear all,
>
> I am wanting to generate in situ FADH2 (the reduced form of flavin adenine
> dinucleotide) from FAD. I need a system that is quite clean as I want to
> test the ease of reduction of a series of organic compounds in the presence
> of the FADH2 and do not wish for too many complicating species. I have seen
> general references to use of NADH/NADPH and use of NaBH4. If anyone has
> specific conditions that can be used that would be appreciated. Also
> suggested assay methods for checking the activity of FAD would be useful.
>
> Thank you
>
> Joanne
> _________________________________________________________
> Dr Joanne Jamie Phone: (+61) (2) 9850 8283
> Deparment of Chemistry Fax: (+61) (2) 9850 8313
> Macquarie University E-mail: joanne.jamie$##$mq.edu.au
> Sydney, NSW, 2109
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