I have a proteolytic enzyme activity that is inhibited by EDTA, suggesting that the enzyme I'm investigating is a metalloprotease.
Unfortunately, EDTA can chelate several divalent cations, including Ca2+, Zn2+, Mg2+, and Mn2+, which are arguably the most common ion cofactors used by metalloproteases.
In the Sigma product literature, EDTA is listed as only targeting Ca2+ and Zn2+, and that to discriminate between Ca2+ and Zn2+ effects, to use EGTA and 1,10-phenanthroline (respectively). Nothing is mentioned about Mg2+ or Mn2+.
1) Does anybody agree that EGTA (Ca2+-"specific") and 1,10-phenanthroline (Zn2+-"specific") are the best chelators to use to distinguish between Ca2+ and Zn2+ effects?
2) At what concentrations?
3) Are there any better (more specific) chelators to use?
4) What about something specific for Mg2+ or Mn2+?
Thank you in advance to any fellow scientists that may have some helpful advice/tips to offer!
Cheers,
Salim
I'm under the impression that EDTA should be able to strip Mg2+ and Mn2+ as well. Why can't you just strip everything with EDTA, pass your sample through a desalting column, and titrate the metals back in? That way, you have a better control on the concentration of the metal ions.
Arthur makes a good point, removing all metals and then putting a specific one back is a 'cleaner' approach.
Nevertheless, have a look at the table for chelators and their affinities:
http://www.dojindo.com/Images/Product%20Photo/Chelate_Table_of_Stability_Constants.pdf
If you want to be sure about your concentration, mix the metal salt with EDTA to clamp the concentration, most biological systems are really sensitive to Ca2+ and your impurities from your buffers are sufficient to get in the right concentration to trigger physiological responses.
Don't be surprised if it turns out your protease is not completely selective for a particular metal ion. I worked on methionine aminopeptidase from bacteria. It can use Mn2+, Co2+ or Fe2+.
If you are trying to identify the metal that might be present in vivo in the enzyme, you might try atomic absorption spectroscopy. Assuming you can purify enough of the enzyme, AA can directly report metal content in most cases. The issue may be getting enough pure enzyme to provide for AA.
I agree that many ions could bind and support enzyme activity. That might make something like AA, an analytical chemistry approach, a bit more accurate than the biochemical approach of stripping with a chelator and trying to 'refill' with other ions.
I have a similar problem. I work with a toxin, which activity is abolished by using EGTA or EDTA. Here is a really detailed article about EDTA. I found it really useful in understanding how EDTA works and how non-specific it is indeed:
http://plymsea.ac.uk/1894/1/The_chemistry_of_ethylenediamine_tetra-acetic_acid_in_sea_water.pdf
We used DTPA (extracellular zinc chelator). It is thought to be quite specific.
Good luck
About sequestering agents for magnesium(II): besides the above suggested EDTA (*), you may alternatively consider either pyrophosphate or polyphosphates.
(*) Cf. (e.g.): http://www.titrations.info/EDTA-titration-magnesium
About sequestering agents for zinc(II): we may consider ammonia, BAL (2,3-dimercapto-1-propanol), EDTA (ethylenediaminetetraacetate), gluconate, lactate, picolinate and TETA (triethylenetetramine); cf.:
https://www.researchgate.net/post/Other-Zn2-complex-in-water
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