I am working with vanadium dependent bromoperoxidase which has vanadium in its active site and (presumingly) several magnesium ions attached which do not take part in the catalytic reactions of the enzyme (presumingly, again). I wish to remove the magnesium selectively from the enzyme while preserving the vanadium in the active site, i.e. the haloform of the enzyme. Now, EDTA is obviously too aggressive and will chelate nonspecifically. Is there any other alternative that would exclusively target magnesium?
Remove both with edta, run ultrafiltration and then add just vanadium
As Omar said, remove both metals with EDTA and add in the vanadium. An alternative to ultrafiltration would be dialysis. So add your EDTA to the protein to chelate all your metals, remove the EDTA by dialysing against your buffer without EDTA and then dialyse in the Vanadium.
The above protocols would do it if the V was a simple metal ion. But the true coenzyme is a pentavalent form of Vanadate (N-VO4-), which happens to be linked by a covalent coordinated N-V bond. Dialysis may actually wash out the Mg2+ effectively, and EDTA may also do the job, as the
EDTA + EnzN-VO4- ---> EnzN + EDTA-(C2H4O2)2N-VO4-
is unlikely to happen, because the tertiary amines of EDTA present significant steric hindrance.
In addition, you could try some other chelating agents, such as imidazol, which may imitate the HIS-V componud in the enzyme and is more likelly to remove the VO4- coenzyme (if that suits your needs), or you may try pyrphosphate or citric acid, which are even less likely than EDTA to displace Vanadate and can form pretty stable (though reversible) Mg2+-complexes.
May I suggest you to dig a bit deeper into any subject before you send out you question? - Remember that not everybody knows your enzyme as well as you do, and most of us have limited time to answer questions, so we are unlikely to investigate the system in-depth before offering advice. I just happened to know a bit about that enzyme.
Have they been give high quality information to begin with, I am sure Sam's and Omar's advice would have been even more focused and even more helpful to you.
Best wishes
Rogelio
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