[RASMB] time to reach equilibrium

Fri Jun 6 15:43:00 PDT 2003

Seldom do I disagree with Jack, but slow association kinetics, even for
small oligomers, is much more common than people realize. 

One published example is Genentech's anti-VEGF antibody (coincidentally the
one that's driving up their stock price lately). That makes reversible
dimers, but the time to dissociate is hours to days depending on solution
conditions. Moore et al. (1999) Biochemistry 38, 13960-13967. 

Based on our experience nearly all antibodies do this to some extent, and
often even more slowly than that one. (Antibodies make covalent oligomers
too). 

I've seen similar phenomena in other proteins also. Just the other day I
worked on a system that appears to be primarily dimer-tetramer (very tight
dimer), and to my surprise you can resolve dimer from tetramer in
sedimentation velocity and get proportions consistent with the sedimentation
equilibrium data. This is quite a small protein too.

The good news is that velocity is a great way to detect these long-lived but
reversible oligomers. Sorry, this isn't published stuff, and I can't tell
you what this dimer-tetramer system is, but believe me, if you look closely
this phenomenon is not rare.

Just why the kinetics are so slow is an interesting question. Presumably
that indicates association is linked to a significant conformation change
(maybe exposure of hydrophobic residues?). These processes probably have
very high activation energies, so as others have pointed out, going to low
temperature may make equilibration much slower.

I can't say to what extent these phenomenon are a common cause of slow time
to equilibrium, but in my opinion the potential is certainly there.

John Philo
Alliance Protein Laboratories

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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 12:01 PM
To: schubert at biophysik.uni-frankfurt.de
Cc: <
Subject: Re: [RASMB] time to reach equilibrium

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Any discussion of time to equilibrium should of course reference the
original paper by vanHolde & Baldwin  1958 J Phys Chem 62: 734-743 where an
equation was given that predicts the time for simple systems.

The question of failure to reach equilibrium cannot easily be separated from
irreversible processes like denatureation/aggregation possibly involving
disufides.  In my experience aggregation during the runs is the primary
reason for failure in reaching a true equilibrium.  This assignment must be
combined with fitting efforts - can the data be globally fit to a descrete
model or are separate K's for each loading concentration required.  

Often lower temperature helps to achive an approach to equilibrium that is
interpretatble but clearly involves aggregtaion events - see S.S. Rosenfeld,
J.J. CORREIA, J. Xing, B. Rener and H.C. Cheung (1996) "Structural Studies
of Kinesin-Nucleotide Intermediates", J. Biol. Chem., 271: 30212-30221 - in
this case sed vel was invaluable in the final assignments.

If disulfides are a problem that reducants help and in limited cases
stronger reductants like TCEP help more - see J.J. CORREIA, B.M. Chacko,
S.S. Lam and K. Lin. (2001)  "Sedimentation Studies Reveal a Direct Role of
Phosphorylation in Smad3:Smad4 Homo- and Hetero-Trimerization."
Biochemistry, 40, 1473-1482.  The problem is as mention earlier disulfides
often shuffle and reductants just increase the rate of this process without
removing the aggregation.  For Smads they work pretty good - but not
perfectly (see paper above & manuscript in prep).

A final assignment of the cause of the problem requires fitting models that
take into account the nature of the aggregates - cell to cell variation
makes unique assignments difficult.  This was discussed in a book chapter by
Yphantis & co-workers years ago - D.A Yphantis, J.J. CORREIA, M.L. Johnson
and G.-M. Wu (1978).  "Detection of  
Heterogeneity in Self-Associating Systems," in "Physical Aspects of Protein
Interactions," N. Catsimpoolas, ed., Elsevier, pp. 275-303.  A down loadable
version is on my web page under links of interest.  A reinvestigation of
this will appear in Biophys Chem soon.

The suggestion that kinetics of association or that polymerization in
general is the problem with slow equilibrium achievement is not in my
opinion correct.  I am aware of systems like TMV capsid or some tubulin or
actin assembly systems that might exhibit slow kinetics, but the problem is
the mechanism of assembly, indefinite processes that re-equilibrate slowly
due to cascades of assembly/disassembly.  the microscopic events are fast.
In general simple systems like monomer-dimer or 1-3 or 1-2-4 will "never"
exhibit this problem.  If examples in the liturature exist I would like to
hear about them?  I would guess that just like sed vel, if the time constant
for re-equilibration is less than about 100 secs then the boundary will
reflect equilibrium behavior. - Refer to Cann & Kegeles references from 60's
& 70's to assess this.

Another issue I am not sure has been extensively investiagted is that is
there is appreciable monomer around, the sed equil system will equilibrate
fast due to the rapid radial diffusion of the momomer, with the equilibrium
at each radial posistion occuring quickly by mass action.  The equil work by
RC Williams on sickle cell hemoglobin demonstrated this (I need to search
for this ref but its early 70's I think.).

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