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A discussion of the pH dependence of the Kd for calcium of BAPTA-based
fluorescence indicators can be found in:
Kawanishi, T., A.-L. Nieminen, B. Herman and J.J. Lemasters (1991)
Suppression of Ca2+ oscillations in cultured rat hepatocytes by chemical
hypoxia. J. Biol. Chem. 266, 20062-20069.
As a specific example, the Kd of Fura-2 for calcium increases 29% as pH
decreases from 7.07 to 6.61. The log change of Kd which should be
similar for various BAPTA-based indicators of more or less similar
binding affinity can be estimated from Fig. 12.
John J. Lemasters, MD, PhD
Professor and
Director of Cell and Molecular Imaging
Department of Cell and Developmental Biology
University of North Carolina at Chapel Hill
CB# 7090, 236 Taylor Hall
Chapel Hill, NC 27599-7090 USA
Tel: 919-966-5507
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Mario wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Sarah,
>
> I cannot provide you with a definitive answer, but seems to me that
> you could run a quick and dirty trial using hydrolyzed Fluo-3 with
> slides using 1 uM Fluo-3 in buffers that range from 5.0, 6.0, 7.0,
> 8.0, and 9.0 (don't use amine containing buffers). Then you can
> always choose some point of departure from the MP website:
>
> http://www.probes.com/servlets/product?region=Select+Region&item=14218
>
>
> I have to question your statement:
>
> "Therefore, the fluorescence due to Calcium binding at any given
> concentration will be lower at a lower pH, presumably."
>
> When calcium is bound to EGTA and its cousins, protons are often
> kicked off owing the electrostatic effects. In fact, the generation
> of protons is a means used to measure the kd of many carboxylate
> chelators. Thus, I would be a bit surprised if the fluorescence would
> change differently when the calcium is actually bound. The kd will
> change with pH, but the signal generated by bound calcium shouldn't
> change much if at all.
>
> Let us know what you find,
>
> Mario
>
>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Hi-
>> Just wondering if any of you have an idea about the pH dependence of
>> fluo-3 fluorescence. I know the Kd goes down as a result of getting
>> closer to the pKa of the chelator (i.e. the on rate increases).
>> Therefore, the fluorescence due to Calcium binding at any given
>> concentration will be lower at a lower pH, presumably. However, what
>> I'm searching for is whether protonating one of the chelating groups
>> will cause an increase in fluorescence on its own. I'm waiting on an
>> article about this from ILL, but thought I'd try you all too...
>> Thanks-
>> Sarah
>>
>> ---------------------------------------------------------------
>> Sarah Locknar, PhD
>> Director, Neuroscience COBRE Imaging Core
>> University of Vermont
>> 802-656-0413
>> ---------------------------------------------------------------
>
>
>
> --
> _________________________________________________________________
> Mario M. Moronne, Ph.D.
> NanoMed Technologies
> FAX (510) 528-8076
> 1561 Posen Ave
> Berkeley, CA
> 94706
>
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