[RASMB] disulfides

[Holger Strauss]

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)
Robert-Roessle Strasse 10

13125 Berlin/Germany

Tel: +49 (0)30 94793 - 223 (office)
                     - 316 (lab)

Fax: +49 (0)30 94793 - 169 


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Science is spectrum analysis; art is photosynthesis.

                                                    Karl Kraus