Hey guys I'm trying to isolate a tightly but not covalently bound co-factor from an enzyme I'm working with. I'd like to do it without reducing the co-factor if possible. Is there any mild denaturing protocols I could use?
Thanks guys
Harry
There are many ways of removing the noncovalently bound cofactor by denaturing or destroying the protein. The include proteolytic digestion, chaotropic agents such as urea and guanidine HCl, heat, trichloroacetic acid, perchloric acid, SDS, etc.
If you want to preserve the structure of the protein, then a reversible denaturation method is needed. You might try identifying the minimal concentration of urea needed to release the cofactor (something like 1 or 2 M), then remove the urea by dialysis to allow the protein to refold. This is tricky, though. Unless it is quite dilute, the protein is likely to aggregate or precipitate when partially denatured.
Use urea as stated by Adam, adsorb the protein on an imac column, remove the cofactor, refold the protein still adsorbed, and desorb the folded protein.
It would be better if you specify the protein and the cofactor.
I once had to prepare nucleotide-free Hsc70, a heat shock protein that binds ATP or ADP very tightly (2 weeks dialysis against activated charcoal would not remove them!). The solution was to remove the bound Mg with EDTA to weaken the bond and then precipitate the protein with ammonium sulphate. This denatured the protein enough to release most of the nucleotide. The protein was still active after this procedure (after addition of ATP, of course).
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