calcium removal from calmodulin

[Gabriel Akopian]

For some time now, our research group has been trying to remove
calcium bound to calmodulin (4 moles Ca2+/1 mole of CaM), using 
a chelex-100 column.  Using a sodium bicarb and sometimes ammonium 
bicarb at neutral pH (ranging from 7.0 to 9.0) we've managed to 
only remove three moles of Calcium per mole of calmodulin.  However,
when the column is not equilibrated with buffer (pH 10.0 to 12.0) all
for moles of calcium are removed.  The only problem is that at this 
pH range our protein is denaturing...Most likely, the hidden Ca2+ 
binding site is being exposed (after denaturation) and the calcium is
being chelated.  Has anyone ever worked with calcium removal from 
calmodulin or any of the other calcium binding proteins.  Also, 
extensive dialysis with EGTA or EDTA doesn't help...we always have 
one mole of Ca2+ for every mole of Calmodulin.