The answer depends on a number of questions:
* What metal ion are you trying to eliminate?
* What kind of metal-binding site is involved?
o A peripheral or loose binding site? (e.g. surface calcium
ions)--these may respond to chelators
o An active site coordinated metal? (e.g., metalloenzyme)--these
can be refractory
Many metalloenzymes are not going to give up their metal to chelators,
or just any chelator, or at all. Denaturation, dialysis, and refolding
is an extreme way of removing metal ions to make apoprotein. Won't work
for every protein. Chelation can be highly specific, that is one
chelator may work, while another, similar one, will not.
Some metal ions are notoriously difficult to eliminate, because they are
adventitious trace contaminants in nearly everything, e.g. zinc and
maybe even iron. (Plastic-ware seems to be often loaded with trace iron,
and also is capable of adsorbing metal ions form solution.) To make
apo-enzymes from zinc proteins, you have to go to heroic efforts to
ensure that glassware, water, buffers, and reagents are zinc-free,
especially if you don't have high (mM) concentrations of protein to work
with.
A His-tag is very likely to snag adventitious metals from solution, and
can often mess up metal analysis for metalloproteins by providing
"extra" metal. If this is a problem for your application, you may want
to consider removing the His-tag.
If you are making apoenzyme to get a different metal installed
(metallosubstitution), there are slightly easier ways to do that than
going through the apoenzyme route.
Cheers,
_______________________________________
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: [email protected]
On 5/19/2014 1:20 PM, SUBSCRIBE CCP4BB Adam Brummett wrote:
Hello All,
I apologize for the non-crystal related question. I am trying to get a fully
metal-free apo enzyme. The 6x His construct is consistently purified with some
metal (20-30%). I have attempted chelating away the metal with up to 30 mM EDTA
and DFO and then dialyzing it away, but this has shown little to no effect. Any
thoughts or recommendations would be greatly appreciated. Thanks.
Adam
