Rf: EDTA in cell fractionatio

Curt Ashendel ashendel at aclcb.purdue.edu
Wed Nov 2 16:01:12 EST 1994


On 2 Nov 1994 15:42:04 -0000, 
LOGAND  <logand at msdos.montpellier.inra.fr> wrote:

>>2nd Year requires background of using disodium EDTA in cell
>>fractionation.  Is it just to buffer Ca2+ ions?
>
>>Just a quick note of why the EDTA is used and typical concs. would be
>>gratefully appreciated.  Send clues by email, thanks.
>
>>Neil Benson
>
>It could be for at least two reasons:
>
>1. to buffer magnesium and calcium cons. so conc. too low for activation of 
>proteases or other detrimental enzymes.(EDTA has higher affinity for Mg, EGTA 
>for Ca)
>

This reason stated above for its use is essentially correct. I include EDTA 
to reduce the level of free Ca ions (primarily released by lysis from 
sequestration in the mitochondia), so that [Ca++] < 10^-10, and hence too 
low to activate calcium-dependent proteases, such as the aptly named 
calpain. 

I think that Mg has virtually no effect on proteases, although it is 
needed for most phosphatases and most DNases.

However, the last statement above, that "EDTA has a higher affinity for Mg 
and EGTA has a higher affinity for Ca," is not completely correct and is a 
myth that perpetuates. If you look at the original EGTA paper 
(Schmid and Reilley, Analytical Chem.  29: 264, 1957), the log Kd values 
for each ligand and chleator at pH 8.0 are approximately:

       EDTA        EGTA
Ca++   -10.5       -10.5

Mg++    -8.7        -5.2

The correct statement is that EGTA has an affinity for Magnesium ions that 
is lower than the affinity of EDTA for Mg++.  EGTA should be used when 
it is desired to have less impact on [Mg++] than on  [Ca++]. (This often is 
the case when isolating membranous subcellular fractions or when it is 
important to keep the nuclei intact.)

EGTA and EDTA have equal affinities for calcium over their effective pH 
ranges.  Thus, one rarely really needs to add both EDTA and EGTA as one or 
the other should suffice.  Also, neither agent allows for creating a 
condition of complexed magnesium in the presence of excess free calcium.

Curt Ashendel
Purdue University
West Lafayette, IN
ashendel at aclcb.purdue.edu



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