Could those who responded with numbers for affinities of imidazole for metal
ions please divulge their sources? It is not that I doubt their veracities,
but it would be a nice reference to have on hand.
For those wondering about why I was asking about imidazole's affinity for
metal ions, I was wondering whether the presence of imidazole would affect a
metal-ion-dependent reaction. With, for example, 200 mM imidazole and 10 mM
Ca++ or Mg++, what would be the amount of free metal? This can of course be
calculated from imidazole's binding constant for these ions, which is
another reason I ask for the sources of the numbers quoted in a couple of
the responses.
Thanks for all of the helpful responses so far,
Jacob Keller
*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [EMAIL PROTECTED]
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----- Original Message -----
From: "Nadir T. Mrabet" <[EMAIL PROTECTED]>
To: "Jacob Keller" <[EMAIL PROTECTED]>
Cc: <[email protected]>
Sent: Friday, July 18, 2008 8:55 AM
Subject: Re: [ccp4bb] Imidazole's ability to chelate metal ions
Imidazole can indeed complex (monodentate) metal ions but not chelate them
(bidendate, at least).
However, the stability constant, K, of such complexes is rather low, eg
log K = 0.1 for Mg, 3.3 for Fe and 4.2 for Cu.
In comparison, metal chelates are formed with EDTA, for which log K = 10.6
for Mg, 14.2 for Fe and 18.8 for Cu.
So the difference amounts to several orders of magnitude.
It should also be pointed out that the competitive effect of imidazole in
IMAC does not involve binding to free metal ions,
but instead coordination to immobilized metal chelates, eg
Ni(II)-nitrilotriacetate (Ni-NTA, where NTA is the chelator).
In any situation where one assays a protein whose activity and/or
stability and/or else is/are metal dependent, one should
rather use buffers (see below) that do not interfere (eg Good's buffers).
I suspect the imidazole in your case is either a buffer (pKa 7.0) or else
results from competitive elution from an IMAC column.
What should be done depends on your exact conditions.
hth,
Nadir
--
Pr. Nadir T. Mrabet
Cellular & Molecular Biochemistry
INSERM U-724
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax: +33 (0)3.83.68.32.79
E-mail: [EMAIL PROTECTED]
Jacob Keller wrote:
Dear Crystallographers,
Does anybody happen to know whether imidazole is able to chelate metal
ions in solution? It seems reasonable that since it can compete for
binding to IMAC resins, it should have some affinity for at least Ni++
and Co++, but what about metal ions like Ca++ and Mg++? I assume that the
affinity is weak, but at the concentrations at which we are wont to use
it in our elutions (~100-500 mM), does it not seem likely that other
metal ions are being competed away from our proteins as well?
Jacob Keller
*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]>
*******************************************
