[RASMB] disulfides

[John Philo]

Holger,

I would think the best way to measure your fraction of disulfide-linked
dimer might be to run sedimentation velocity at a very low concentration,
where the non-covalent dimers are essentially absent. One could also, of
course, try to quantitate the staining of monomer and dimer bands on
non-reducing SDS PAGE.

In principle you could actually purify away your disulfide-linked dimer via
size-exclusion chromatography at a very low protein concentration. 

It also would not be surprising to find that the disulfide-linked dimer
could be separated on reverse-phase HPLC. That might not work as a
purification scheme, since I don't know whether your monomer would be easily
refolded after such a purification, but if you get a good separation you
could at least get good quantitation of the dimer fraction from integrating
the peaks.

John Philo

-----Original Message-----
From: rasmb-admin at rasmb-email.bbri.org
[mailto:rasmb-admin at rasmb-email.bbri.org] On Behalf Of Holger Strauss
Sent: Wednesday, June 04, 2003 6:33 AM
To: mail-to-all at rasmb
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

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - 

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

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