Calcium removal from Calmodulin

Gabriel Akopian akopian at oxy.edu
Thu Mar 30 19:42:25 EST 1995


Can anyone, someone, please.....please....send me suggestions on how
to remove calcium from calmodulin.  We've been using a chelex-100
column (1cm x 20cm) in all sorts of buffers (ammonium bicarb, sodium 
acetate) at various pH levels (from 7.5 to 9.0).  At these buffered 
pH ranges we've only managed to remove 3 out of the 4 Ca2+ that bind
to calmodulin.  When we don't use any buffers, (pH of chelex 10 to 12)
we can remove all four Ca2+.  The only problem is, that at this pH
range, the protein begins to denature (based on UV spec).  My guess is
that at this pH the Ca2+ binding site that refuses to let go of the
Ca2+ becomes exposed (because of denaturation) and thus can readily
give up the last Ca2+ to the chelex.  Does anyone have any ideas....
Also, all metal ion determinations are done by Atomic Absorbance 
measurements.



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