GUS assay with substrate MUG - is there oxidative degradation of MU?

Wolfgang Schechinger hubahopp at gmx.de
Thu Mar 18 18:58:46 EST 2004


Hi Dirk, 

of course, when you blast it with UV, you'll finally kill it. Easiest to
test your hypothesis about oxidation is setting up a control experiment
with MU. Maybe your plant extract does some harm to it?

What you observe might be some sort of quenching,  depending on other
reactions - "the metabolic status" - of your extract and some
thermodynamics of fluorophor-quencher-interactions. Could be due to
(hydro-)quinones in your extract. You could add some albumin or salt or
radical scavengers (eg vitamin C and E) to improve the stability.

Are you aware of other substrates like Fluorescin-bis-glucuronide,
PFB-FDGlcU, ELF-Glucuronide (all available from eg. molecular probes, in
Germany: Mobitec) or good old p-nitrophenyl-glucuronide (you'll need a
photometer for the latter and you'll have to alkalize with NaOH, so it's
destructive)? 

As a third reason comes into my mind (assuming you work at constant
temperature which otherwise also might affect the fluorescence) that what
you observe maybe are in fact binding kinetics (all released MU is absorbed
first, so you'll see a decrease first, then later you'll see the real
production. In that case, your V shaped curve would be the superposition of
a binding curve and the hydrolysis curve. In this case, a pre-incubation in
the cold (where the enzyme does not work but thermodynmics only see 270
instead of 300 Kelvin which does not matter so much to kinetics) and / or
pre-treatment of your microplates with albumin, gelatin, or even starting
with some free MU added to your assay might solve your problem. Controls
without extract or fluo are advisable. Discrete spectra of your RxN might
reveal insights too as your blues might be just the background fluorescence
in the worst case.

Finally, to simplify things, is it possible to record a background curve
and subtract it from your data?

Wo

At 17:31 18.03.2004 +0100, Dirk Müller wrote:
>I have a severe problem with my GUS assay. I measure proteinextract
>from fungal cells, expressing b-glucuronidase with the substrate MUG.
>The b-glucuronidase converts MUG to the fluorophor MU by removing the
>glucuronide. The fluor, eszence ist measured with a microtiter plate
>reader at the appropiate wavelength. In nearly all samples I observe a
>signal reduction from time point 0 min to 20 min and then an increase
>for over night incubation. The curve looks like a "V". The microtiter
>plate is not sealed between the first two time points; for over night
>incubation we seal it with a special foil. Is it possible that oxygen
>(or light) damages the MU in a way that it looses its ability to act
>as a fluorophor at the normal wavelength? In the over night reaction
>the plates are sealed from light and oxygen so that glucuronidase can
>again accumulate MU from MUG without "destroying" the MU?
>
>I hope someone can help me or has observed a similar effect - this is
>really a pressing problem.
>
>thank you, Dirk...
>
>
Wolfgang Schechinger


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