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anaerobic nitroreductase assay on PAGE

Mary Elyse Lucero malucero at nmsu.edu
Mon Jul 25 16:32:16 EST 1994


	Rafii et. al. have described a method for assay of nitroreductase
activity which involves running cell extracts on a nondenaturing anaerobic
polyacrylamide gel.  The gel is then incubated anaerobically with a
solution containing nitrobenzoic acid, FAD, and NADH; followed by
incubation in a solution containing trichloroacetic acid and sodium
nitrite.  Ammonium sulfamate, and later, N-(1-Naphthyl) ethylenediamine
dihydrochloride (NEDD) are then added, and the NEDD reacts with the amino
groups (reduced nitro groups) to form a reddish colored band where the
nitroreductase is located. 

	Has anyone tried this method?  I am curious as to the sensitivity
of the method (How much protein must be present in order to obtain a
visible band?), and in whether or not other nitroaromatics would react as
readily with the NEDD as does nitrobenzoic acid.  (I am looking at enzymes
involved in reduction of Trinitrotoluene)

	I would also like suggestions on maintaining anaerobic conditions
throughout this procedure, since this was not elaborated on in the paper I
read.

	The procedure is described in "Reduction of Nitroaromatic
Compounds by Anaerobic Bacteria Isolated from the Human Gastrointestinal
Tract" by Fatemen Rafii, Wirt Franklin, Robert Heflich, and Carl Cerniglia
in Applied and Environmental Microbiology, Vol. 57 #4, 1991. I am waiting
for interlibrary loan to procure another paper by the same group, which
appears to describe the assay in further detail, but I would still
appreciate any tips, suggestions, or comments on the method. 

Thanks!


Mary Lucero
malucero at nmsu.edu	





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