[RASMB] disulfides

[JA KORNBLATT]

this is not an easily solved problem for at least a minimum of two
reasons:

disulfides, in the presence of free -SH are not stable, they undergo
disulfide exchange. that means that you have dynamic equilibria during
the centrifugation but that you do not know the rates at which exchange
is going on.

you have five -SH. the disulfides can, in principle, form between any of
the pairs. would you be able to detect these by ms, dsc or cd? i wouldn't
think so.

if i were you i would reduce them all and alkylate them so as to obviate
the problem.

all the best
jack kornblatt



On Wed, 4 Jun 2003, Holger Strauss wrote:

> Date: Wed, 04 Jun 2003 15:32:53 +0200 (MEDT)
> From: Holger Strauss <strauss at fmp-berlin.de>
> To: mail-to-all at rasmb <rasmb at rasmb-email.bbri.org>
> Subject: [RASMB] disulfides
>
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>
> Hello all,
>
> there is the following situation: a given protein, 20 kDa, recombinantly
> expressed in e.coli, 5 free cysteines, one band in SDS-PAGE/Coomassie (no
> silver stain done), 2 species in the MALDI-spec, monomer and covalent
> dimer, nice CD and melting curve. The buffer is Tris pH 7.4, 100 mM NaCl,
> 5 mM beta-Mercapto.
>
> OK, there seems to be a portion of specific disulfide linkage, and the
> system slighlty self-associates (point-average Mw, integral
> c(S)/concentration all increase), but it's close-to-impossible to describe
> the equilibrium gradients by a simple standard model, which is due, I
> think, to a significant contribution of the covalent linked dimer to the
> measured concentration distribution.
>
> So, here are my questions:
>
> - if one could measure the fraction of covalent dimer, one could simulate
> its equilibrium distribution, and substract it from the experimental
> gradients; how would one measure this precisely?
>
> - if it can't be measured precisely, maybe one could fit directly for the
> fraction of covalent dimer? (which programm would allow you to do this?
> How to judge the reliability of this number?)
>
> - (that's more general): what does the term "incompetent monomer" mean on
> a structural basis (assuming that folded equals competent)? (Treating such
> a thing would be very much the same as with a covalent dimer, I presume?)
>
> Greetings to all, Holger
>
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>
> Holger Strauss
>
> Forschungsinstitut fuer Molekulare Pharmakologie (FMP)
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>
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> Science is spectrum analysis; art is photosynthesis.
>
>                                                     Karl Kraus
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