Calculating free Ca2++ with EGTA buffers

CZJ at CU.NIH.GOV CZJ at CU.NIH.GOV
Thu Sep 10 08:29:12 EST 1992


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> From: Fergus_Doherty at vme.ccc.nottingham.ac.uk
> Subject: Calculating free Ca2++ with EGTA buffers
> Date: 10 Sep 92 10:00:35 GMT
> Sender: list-admin at daresbury.ac.uk
> Original-To: BIO-SOFT at uk.ac.daresbury
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> Can anyone help me to calculate free Ca2++ in EGTA/CA buffers?  Basically
> I want to determine the effect of Ca on enzyme activity, and relatedly
> translocation of an enzyme to membranes-which is Ca -dependent.  I need
> to include EGTA to completely inhibit the enzyme, and then add back Ca.
> This must have been done thousands of times but I am completely
> bamboozeld by the books I've looked at.  What I need is a simple
> explanation and perhaps a program, spreadsheet or Hypercard stack
> for the calculations (so Mac, or PC Excel).  Thanks.
> FErgus Doherty, Biochemistry, Nottingham University, UK.
>
>

The calculation is straight forward.  You will need the stability
constant for the Ca2+ EGTA complex and the ionization constants
for EGTA.  There used to be a book entitled "Stability Constants"
which contained a complete listing of such.  Check with your
Chemistry Department Librarian regarding current sources.  You
should select constants obtained under the same conditions as
your experiments.  Otherwise you will have to extrapolate them to
the appropriate ionic strength using the Debye-Huckel equation.
Once you have the correct constants it a simple matter of writing
down the laws of mass action nad using a little algebra to obtain
the free Ca2+ concentration.  Since it will be small, several
approximations can be used to simplify the algebra.

Jim Cassatt





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