Dimer->monomer->dimer

J. Martinez-Irujo jjmirujo at unav.es
Sat Mar 4 05:20:10 EST 2000


"Lars H. Østergaard" wrote:

> Hi everybody
>
> Any ideas on how to monomerize a dimer (hydrophobic interaction) - and

> then afterwards
> being able to form an active dimer again? Would a drop in
> salt-concentration in the buffer
> do the job? Or a mild detergent? Any suggestion would be gratefully
> appriciated.
>
> Thanks
>
> Lars

In fact, it depends on the strength of the interaction. Try to  prepare
your protein in a solution containing different concentrations (5%, 10%,
15%, 20%...) of an organic solvent (methanol, ethanol, acetonitrile...)
in a suitable buffer at 0-4 oC (dilute 1:1 or  dialyze against this
buffer). Remember that the absolute concentration of the protein affects
to the monomer-dimer equilibrium, so test several concentrations of your
protein. Measure  the relative amount of monomer-dimer by gel filtration
chromatography (a high performance column  will be needed to separate
them). You can also follow the process by measuring enzyme activity (a
100-fold dilution may be necessary to avoid solvent effects). To get the
dimer again, dilute (or dialyze) your protein in a solvent-free buffer.
Adding a antichaotropic salt  (try  0,2-2 M NaCl or (NH2)2SO4) will
help. It is advisable not to dilute so much your protein in this step
since the dimer/monomer relation at the equilibrium will be decreased.
On the other hand, if excesive solvent remains after dilution you can
expect some monomer remaining at the end.

Good luck.


--
Juan J. Martinez Irujo

Departamento de Bioquimica
Universidad de Navarra
Pamplona, Spain


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