disulfide bond or dimerization via iron cluster

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Mon Oct 22 19:45:52 EST 2001


Dynamic light scattering would not resolve -S-S- versus -S-Fe-S-. It is the
same dimer, essentially, so from the point of view of DLS it will tumble the
same, have the same gyration radius, etc. etc. I would recommend 1) mass
spectrometry 2) purify your dimer (e.g. size it away from all potential
sources of iron, using new size exchange resin and iron-free buffers) and
look for iron using e.g. AA spectroscopy or conventional chemical methods of
iron determination.

Try reducing your dimer with TCEP (Pierce and many other companies). It
should not affect Fe-S bonds but will readily break -S-S-. Or so I hope (I
may be wrong). Detect using non-reducing SDS-PAGE or any other convenient
technique.

A.G.E.

"Dominic-Luc Webb molmed" <domweb at mbox.ki.se> wrote in message
news:Pine.GSO.4.40.0110222156370.10911-100000 at mbox.ki.se...
> On 22 Oct 2001, Guy Nadeau wrote:
>
>    > hi,
>    > What kind of procedure i should try if i want to determine if my (!)
protein
>    > dimerizes via disulfide bond or via a iron-sulfur like interaction ?
It's
>    > o.k for the DTT (-S-S-) but a specific assay for dimerization of
iron-sulfur
>    > protein should be available somewhere... but i still can't find it.
>
>
> Not my specialty, but a concept I have been exploring for
> a similar problem is use of Dynamic Light Scatter analysis.
> This is a structural biology technique that gives info
> about the globular structure of a protein. I would presume
> it can give clues as to whether or not dimers are formed,
> so maybe you could use this technique +/- iron/sulfur. This
> is simply not my specialty, so I hope an experienced
> structural biologist will comment further.
>
>
>
> Cheers,
>
>
>
> Dominic
>
> Lab:
> Department of Molecular Medicine
> Endocrinology and Diabetes Unit
> Rolf Luft Center for Diabetes Research
> Karolinska Hospital L3
> S-17176 Stockholm
> Sweden
> Tel: Int+46-8-517-74829
> Fax: Int+46-8-517-79450
> Internet Email: dominic at enk.ks.se
> ________________________________________
>





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