When enzymes were eluted with high concentration immidazole, some of them lost their activity in 2 days. I want to know how immidazole affect enzyme or other protein. I can't find article about that.
One may assume imidazole at sufficiently high concentrations will interfere with the activity of enzymes whose catalytic mechanism employs Histidine residues in charge-transfer relay systems - for examples hydrolyzing/esterifying enzymes such as those involved in peptide bond hydrolysis and phosphorylation/sulphation. This is why dialysis before, say, thrombin/fXa cleavage of His-tag purified recombinant fusion proteins is desirable = more rapid and efficient cleavage! Obviously this phenomena will be a second-order phenomenon (dependent on the concentrations of both reactants) so I suggest you do your own steady-state experiments by titrating different amounts of Histidine/Imidazole/Glycine(control) against a fixed concentration of your enzyme and measure initial rates of hydrolysis... From published data on the known activity of your enzyme superfamily it should be possible to rationalize what's going on... Have fun
One possibility is that the protein was unstable due to the pH or to the ionic strength. You can change these by dialysis against a different buffer. Include 20% glycerol as a stabilizer and store the protein in small aliquots at -80oC or in liquid nitrogen.
Dear Adam
I remove high concentration of immidazole by dialysis or I add glycerol 10% in my elution. glycerol cause my enzyme to remain active.
but immidozole doesn't negative effect on another enzyme.
I want to know effect of immidazole on protiens
One may assume imidazole at sufficiently high concentrations will interfere with the activity of enzymes whose catalytic mechanism employs Histidine residues in charge-transfer relay systems - for examples hydrolyzing/esterifying enzymes such as those involved in peptide bond hydrolysis and phosphorylation/sulphation. This is why dialysis before, say, thrombin/fXa cleavage of His-tag purified recombinant fusion proteins is desirable = more rapid and efficient cleavage! Obviously this phenomena will be a second-order phenomenon (dependent on the concentrations of both reactants) so I suggest you do your own steady-state experiments by titrating different amounts of Histidine/Imidazole/Glycine(control) against a fixed concentration of your enzyme and measure initial rates of hydrolysis... From published data on the known activity of your enzyme superfamily it should be possible to rationalize what's going on... Have fun
Hi, what protein are we talking to? Imidazole could act an a proton transfer in some cases and it could inactivate your protein...If your protein behaves well you can try to purify the protein in the unfolded state adding urea in the system (check the Qiagen manual), remove the imidazole and finally refold your happy protein.
Hi there,
I think instead of trying to understand how imidazole is affecting protein activity/stability in general, you should systematically remove it right after NiNTA as this compound could interfere with downstream steps of your purification/studies... Except if imidazole is relevant towards your protein biological role.
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