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How does urea inhibit enzymes?
2014-12-10 16:12:06
Urea is a very strong denaturing reagent because of its highly polar structure (H2N-CO-NH2, with a double bond between C and O).
The strong localized charges will interact with the aminoacid structure of any protein and enzyme (especially the secondary chains) and disrupt their function. So it is not directed to the active site.
Theoretically, the process is reversible, but you need to progressively remove UREA from your solution, as if you remove it completely the denatured proteins will precipitate as insoluble material.
This is what you should do: resuspend your samples in urea (generally between 6 and 8M, but less could also be enough) to solubilize and inactivate the enzyme. Then dialyse your protein suspension against the same buffer with half the amount of UREA you started with (e.g 3M). After about 1 h, you can start changing dialysis buffer and serially diluting your UREA until about 0.5M or less and then no urea at all (so, 6M, 3M, 1.5M, 0.75M, 0.375M, 0M). Each step for about 1-2 hours. I wouldn't dialyse for too long as the proteins can go out of solution.