I previously tried some protocol to remove metal from enzyme to make apoenzyme, but it did not work.
Questions in this regard:
1. My apoenzyme gave 20% activity but in many literary sources their apoezyme gave less than 5% activity, how is it possible and why?
Dear Anurag
which is the metal of your enzime and which aminoacids are involved in its binding?
there is not a Universal Protocool because it depends also from the intrinsic stability of the apo enzime.
in 2 cases for Human cu,zn Sod1 and clostridium Zmp1 for me an incubation in edta 20mM at acidic ph (ph 2.7) foliowed by buffer exchange by pd-10 or dialysys to remove metal and edta worked well but i was lucky because not al the proteins are stable at so low ph.
Of course is essential that your buffer are made with water that do not contain any metal traces otherwise is possible that the apo protein acquire some metal from the Solutions and i not apo anymore.
theoretically at the end of The process you need to quantify the metal bound to your protein by submiting the apo and metallated sampkes to an icp-ms analisys or similar becauses otherwise is it difficult to correlate activity with metal content.. is it possible that 5% of activity of apo is due to the fact that there is still 5% of metallated as well as is possible that your metallated show only 20% of activity because there is some apo it because during the production some other metal bound aspecifically in The site and The protein is not active.
Hi there,
In case of metal cation, NTA-agarose may be used as a strong cation exchanger:
Article Removing coordinated metal ions from proteins: A fast and mi...
@Manuele Martinelli Thank you ! But in my case, Zn is the metal and ASP, HIS are the amino acid residues which bound the Zn metal.
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