[RASMB] time to reach equilibrium

[John Philo]

First, just to clarify (though this will be obvious if you read the paper)
the slow, reversible dimerization of antibodies I am talking about is NOT
due to disulfide formation. These are believed to be strictly non-covalent
oligomers. Yes, of course disulfide-linked dimers can also exist, but that
is essentially an irreversible phenomenon. 

Second, no I can't directly link this to slow time to equilibrium or to poor
fitting to a global model. If I see this sort of thing in velocity then I
usually wouldn't try an equilibrium experiment. The recent case I mentioned
of a slow dimer-tetramer system was not drastically slow to reach
equilibrium.

Yes, I agree in general one doesn't expect to resolve individual oligomers
in a velocity experiment---it only works when the kinetics are very slow
(half-times of 1000's of seconds or longer). This is indeed something that
causes confusion, and I get a lot of questions from DCDT+ and SVEDBERG users
related to this issue of when is it reasonable to treat an interacting
system as a mixture. 

Sorry, I didn't mean to imply hydrophobic association is intrinsically slow.
I was suggesting a mechanism like a rare, significant unfolding event that
exposes hydrophobic groups normally in the hydrophobic core. 

-----Original Message-----
From: rasmb-admin at rasmb-email.bbri.org
[mailto:rasmb-admin at rasmb-email.bbri.org] On Behalf Of John Correia
Sent: Friday, June 06, 2003 2:12 PM
To: jphilo at mailway.com
Cc: <
Subject: RE: [RASMB] time to reach equilibrium


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John

If you have data & papers to reference on slow kinetics then disagree all
you need to - every system I have worked on is far more aggregation prone?
Seems that stopped flow needs to get rejuvenated as a complementary
technique!  If there are more papers out there let us know!!!  

I would still caution against the idea that polymerization & slow kinetics
go hand in hand.  Of course I never do equilibrium runs without a
concentration series by sed velocity, so I would see it & maybe skip the
equilibrium run to begin with.

Thus, for global analysis of multiple cells you need to use individual K's?
or can you actually get equilibrium & global fits?  With some systems we
work on the aggregation & heterogeneity is different from cell to cell &
condition to condition, so its not a frozen system. Thus, individual K's
works better.  How to extract the true K is the hard part since it depends
upon the aggregation model too.

The Antibodies are S-S linked & thus that is disulfide cross-linking &
re-shuffling - thus slow kinetics here are due to covalent bond reshuffling
- NO?

I will point out a previous email from Avi on using C(s) and fitting for
monomer-dimer-tetramer referred to MW's as if the species separated into
independent or slowly equilibrating zones in a velocity run.  Very slow
kinetics will given separate zones that can be fit as if they are
noninteracting.  Rapid requilibration (100 secs or less) should never give
separate zones for the polymers & thus should never be analyzed in this way.
This appears to be confusing to some new users.

Entropically driven polymers like tubulin polymers are primarily hydrophobic
& the assembly steps are not slow.  Significant conformational/folding
linkages seems more reasonable as a mechanism. 
So is this a linkage to unstable or unfavorable folding kinetics?  Thus, the
small peptide/protein connection?  Any evidence of this in the coiled-coil
field??


-------------------------------------------------------------------
 Dr. John J. "Jack" Correia
 Department of Biochemistry
 University of Mississippi Medical Center
 2500 North State Street
 Jackson, MS  39216
 (601) 984-1522                                 
 fax (601) 984-1501                             
 email address: jcorreia at biochem.umsmed.edu     
 homepage location: http://biochemistry.umc.edu/correia.html
 dept homepage location:    http://biochemistry.umc.edu/
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