[RASMB] Aggregation AND m-d Kd

John Correia jcorreia at biochem.umsmed.edu
Wed May 30 15:44:34 PDT 2007


Mitra
 
Detection of aggregation or heterogeneity was to my knowledge 1st dealt
with quantitatively in a book chapter:  
DA Yphantis, J.J. CORREIA, M.L. Johnson and G.M. Wu (1978).  "Detection
of  Heterogeneity in SelfAssociating Systems," in "Physical Aspects of
Protein Interactions," N. Catsimpoolas, ed., Elsevier, pp. 275303. 
Recently Yujia Xu published a graphical method for indicating the
presence of heterogeneity and extracting K's from Kapp values. 
Characterization of macromolecular heterogeneity by equilibrium
sedimentation techniques, Biophys Chem 108, 141-163, 2004.
 
In practice it has been noted for sometime now that fitting individual
loading concentrations will give a trend of increasing Kapp with
decreasing concentration - this applies to equilibrium data, a feature
implemented in earlier versions of Nonlin, and more recently to velocity
data.  see:  J. J. CORREIA, C. A. Sontag, W. F. Stafford and P. J.
Sherwood, (2005) *Models for Direct Boundary Fitting of Indefinite
Ligand-Linked Self-Association,*  in Analytical Ultracentrifugation:
Techniques and Methods. (Ed. D. Scott, S. Harding and A. Rowe) pp 51-63.
As David said, when the aggregates pellet there is no problem, just use
the species in equilibrium at the plateau concentrations.  The problem
arises when the heterogeneity is aggregated dimer or inactive monomer. 
Careful analysis and direct boundary fitting can reveal these features
while also extracting the reversible component of the reactions. 
Aggregation during the run is more difficult and favors velocity methods
or changing conditions like temperature, pH, salt or reducing agents to
induce stability.  
 
-------------------------------------------------------------------
 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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>>> <mitrana at mail.utexas.edu> 05/29/07 3:04 PM >>>

Hi Folks
I have a question (s) - how does the aggregation (and maybe the
subsequent loss)
of a protein sample during SV affect the Kd of the monomer-dimer
equilibrium?
Either by direct fitting of the boundary or considering the average
sedimentation coefficient. Is there any way around this?

Mitra
-- 
Mitra S. Rana
Graduate Student
Institute for Cellular and Molecular Biology
2500 Speedway, UT-Austin
Austin, TX-78712
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