Re: Fluorescent zinc chelators
From: Bill Telford (telfordw@box-t.nih.gov)
Date: Mon May 21 2001 - 12:08:48 EST
Hi Kathleen...
Fluorescent calcium chelators can be used to measure cations other than
calcium, but controlling for calcium and magnesium binding makes this
difficult, particularly for low-concentration trace metals like
zinc. If you're courageous ;-), you might want to try a compound
called "Zinquin", a somewhat cell-permeable fluorescent zinc
chelator that is commercially available (Dojindo Laboratories in Japan
and Toronto Research Chemicals in Canada are two suppliers I'm aware
of). Zinquin is an ethyl ester -modified form of its parent
compound N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide (TSQ), a
non-permeable zinc chelator that can be excited with UV or violet light
(Hg lamp, Ar-ion UV or Kr-ion UV or violet lines all work) and has a
broad emission peak from about 460 to 510 nm. Long ago, I used TSQ
to do fluorometric zinc measurements in a cuvette and to label
fixed/permeablized cells for zinc for microscopy. In cuvette
experiments, TSQ fluoresced brightly only in the presence of zinc -
cadmium and silver (similar atomic radii to zinc) showed some
fluorescence, but these metals are not normally found in cells.
Calcium, magnesium, copper and iron did not cause the chelator to
fluoresce. We did some unpublished fluorescence microscopy work
with it to look for zinc localization in fixed lymphocytes - TSQ
precipitated and fluoresced when bound to zinc at physiological pH,
showing sites with high zinc concentrations. I've tried Zinquin
more recently out of curiosity to see if it would label live cells for
both microscopy and flow - with it, we can roughly monitor zinc uptake in
lymphocytes incubated with micromolar zinc concentrations. However,
I haven't done specificity tests on this more permable reagent, nor
established concentration standards. A few labs have used this
compound to measure intracellular zinc levels in lymphocytes and in
spermatocytes by flow (refs), but no one (to my knowledge) has done a
rigorous job of standardizing this reagent as has been done for indo-1,
fura-2, etc. There are potential drawbacks - it is not clear if
these chelators bind to free zinc only, or can also detect zinc bound to
metal binding proteins like metallothionein. TSQ and Zinquin may
also bind to (although not fluoresce with) other trace metals to some
degree, which may cause an underestimation of zinc levels when other
trace metal concentrations are high. Nevertheless, you might want
to give this reagent a try, with the caveat that it has not been as
well-characterized as other fluorescent chelators. A good
opportunity for original technique development, though! I can send
you some data and more info if you are interested.
Regards,
Bill Telford
CCR-NCI-NIH
Some refs...
Andrews et al., Cytometry 21, 153 (1995)
Zalewski et al., J Histochem Cytochem 42, 877 (1994)
Coyle et al., Biochem J 303, 781 (1994)
Zalewski et al., Chem Biol 1, 153 (1994)
At 12:39 PM 5/17/01 -0500, Kathleen Schell wrote:
>
>I have an investigator who wants to measure levels of Zn++ in
his
>cell lines. Has anyone had any experience with Fura-2 that
could
>give us some pointers? We are planning to excite this dye with
a
>multi-line UV and collect the emission at 510. The literature
that
>we have found just uses spectrophotometry to measure this
metal. Can
>we do this?
>Thanks,
>Kathy
>--
>--------------------------------------------------------------
>Kathy Schell
>Supervisor, UWCCC Flow Cytometry Facility
>600 Highland Ave. K4/535
>Madison, WI 53792
>Voice: 608-263-0313
>e-mail: kschell@facstaff.wisc.edu
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