I am working on protein engineering of tyrosinase enzyme. Our enzymes possesses N-terminal Polyhistidine-tag. I'm using Ni-NTA resins and phosphate buffer for it's purification through metal affinity chromatography. Unfortunately NaCl is an irreversible inhibitor of tyrosinase and should be replaced by another salt. I have tested (NH4)2SO4 instead, but it's showing inhibitory effect again. Is there any other salt that NaCl can be replaced with?
What is the reason for having 'salt' there at all?
Do you have any idea how NaCl acts as an 'irreversible inhibitor' of your enzyme?
The concentration of 'salt' is of course also a consideration? What concentrations of NaCl 'irreversibly inhibit' your enzyme? What concentration (if any) do you need for your chromatography?
NaCl is used to eliminate unspecific electrostatic interactions in IMAC.. You can use any salt (not chelating, not reducing) for this purpose. It looks that you can use sodium nitrate for example, since it sould not inhibit tyrosinase(1), but you should review this aspect more than I did
1.Lerner, A. B. Effect of Ions on Melanin Formation1,2. The Journal of Investigative Dermatology 18, 47–52 (1952).
On the other hand, you can try not to use any salt at all and increase imidazole in binding contions to around 5 or 10 mM to avoid inespecifities in binding
Sodium nitrate would be a good alternative. I should test it. Cl ions interact with Cu ions in the active site of tyrosinase in an irreversible manner. It's been written that (NH4)2SO4 has not any inhibitory effect in the same article "Effect of Ions on Melanin Formation".
I have evaluated melanin synthesis in TB medium. Is that possible that dopaquinone or L-DOPA react with (NH4)2SO4?
Simplest solutions first. Are you sure that non-specific 'sticking' is even an issue for your protein on a Ni-NTA matrix. I suggest you figure that out before we come up with more baroque solutions.
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