Problem to find exact extinction coefficient

[nazych]

Hello,

 

I have two proteins which one is wt protein, and one is mutant of the same protein. I should denature both proteins in the same conditions with 5 M urea and do NMR on both of them and they should be exactly in the same conditions.

 

I already used protoparam tool in expasy and found the extinction coefficient of mutant and is smaller than wt, but when I denatured the protein and did NMR on them, the NMR spectrum of mutant dropped about 30%, but based on expectation, it should only drop about 15 percent.

I have a problem to find their exact concentration. Can you please give me a way to calculate extinction coefficient of the mutant and wt.

 

Thanks.