> >On Wed, 30 Sep
> >1998, Doug Hubatsch wrote:
> > Hi All,
> >Anybody know what happens to the calcium and/or the dye after they have
> >bound together and been through a cycle of excitation and emission?
> >Does the dye undergo a physical change (?) which results in the calcium
> >being released?   If so, is the dye 'weaker' during the next cycle?
> >Regards,
> >Doug
> >
> >

>Hi All,
>
>I am not a calcium specialist but if free calcium concentrations will
>change due to release from the fluorochrome, one can state that the
>incorrect amount of fluorochrome is used.
>The principle should be that the fluorochrome concentration must be that
>low that it does not buffer the calcium concentration. It has to be the
>same as with a pH indicator. This indicator, although it reacts with
>protons may not change the net amount of free protons in a significant
>way, otherwise it works as an buffer and not an indicator.
>
>This is also one reason why fluorescent dyes are used. They are visible at
>extreme low concentrations and give a signal without affecting the
>concentration to a significant amount.
>
>What is significant: 1% I guess is reasonable.
>
>Patrick


Hi everyone.  Referring to the question about calcium dye's...

Patrick van Oostveld raises a most interesting point about the buffering
effects of fluorescent indicators.  As he says, they give a signal without
affecting the concentration to a significant amount.  Or at least they seem
to.  It's easy to calculate the buffering power of an indicator, which of
course depends on the dissociation constant (Kd) of the indicator, and the
concentration at which it is used.  From this information we can calculate
the concentration of the ion-indicator complex, and we can then compare
this with the free ion concentration (obtained from the fluorescence
measurements of course) to obtain the buffering power of the indicator
under
those conditions.

Well, lets do this for a typical practical case, and see what sort of
answer we get.  Anyone of a nervous disposition may well wish to stop
reading at this point, as they may find the conclusions unsettling.  I'll
use the Ca indicator fura2 as an example.  The Kd of this dye in the
cytosol is on the order of 1uM, and it is normally loaded to a
concentration of at least 10uM.  It's generally accepted from a variety of
different measurements that the free cytosolic Ca concentration under
resting conditions is around 100nM.  Since this is about 1/10 of the Kd of
fura2, then about 1/10 of the indicator will be binding Ca under these
conditions (more precisely, the ratio of Ca-fura complex to free fura will
be 1/10, but since most of the fura is still in the free form, this
approximation is close enough).  So the concentration of Ca-fura complex is
around 1/10 of 10uM, i.e. 1uM, and our fluorescence measurements are most
likely telling us that the free Ca concentration is around 100nM, i.e.
about ten times LESS than that.

This isn't a 1% buffering, it's a 1000% buffering!!!!  And the figures I
used are conservative.  In practice fura2 is normally used at a higher
concentration than 10uM, and its Kd in the cytosol is probably rather less
than 1uM, so these factors make the situation even worse.  No, the reason
that fura2 seems not to (seriously) buffer the cytosolic Ca, is NOT because
of some phenomenal sensitivity of this indicator, but is instead because it
is competing with many OTHER cystosolic Ca buffering mechanisms.  It's the
fact that we can therefore massively "overload" the cells with fura2, which
makes it sensitive in practice.  Even so, the risk that both the amplitude
and the time course of cytosolic Ca transients can be distorted by the
presence of the indicator is a real and ever-present one!  Although I've
used fura2 as an example, the same problem applies to all the other Ca
indicators, and almost certainly the pH indicators too (similar calcs on
them could be instructive).
If you wish I'd never told you this, then I'm really very sorry.....


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