redeamer alion85 at
Fri Feb 6 07:47:05 EST 2004

>Hi guys, 
>I am sort of undecided on the following issue. Say if I precipitated
>enzyme that has metal cofactor in its structure (internal), should I
add a
>bit of that metal in the reconstitution buffer. Seems like the answer
is NO,
>because acetone should not change protein conformation or cause any
kind of
>unfolding. The precipitating effect is mainly due to aggregation of
>molecules under more hydrophobic conditions in presence of the
>solvent. Right? 

Hi, Emir!

As i understand You mean the TCA precipitation with upcoming cold
acetone wash step? If so then TCA unfolds and denatures protins for
sure, and then when the hydrophobic core of protein exposes to polar
solvent the molecules of proteins do really aggreggate and
precipitate.... i am not sure but the acetone wash step  denatures the
protein further and makes the environment more hydrophobic... if my
line is right thn you would have no metals as a cofactors inside your
denatured structure of protein... then you have to renaturate your
protein in the presense of some detergents with a metals (a

...from the other hand if your cofactors provoke an allosteric
conformational change or enzymatic activation then if your protein
even in 3D structure or actually in 4D (lets not forget
TIME:]jokingly) the chealted metals could be accessible to solvent
(whatever one)...  and anyways metal are required..

thats my point of view...

------------> Drew

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