From: Walter Stafford <STAFFORD@bbri.harvard.edu>
  To  : rasmb@bbri.harvard.edu
  Date: Wed, 12 Jun 1996 13:14:38 -0400 (EDT)

Use of dc_dt method for low molecular weight molecules

In response to a frequently asked question (viz:

>                I would like to know if anyone have used the "dcdt" method
>to calculate the apparent sedimetation coefficient distribution for
>proteins of low s values ( 0.5S to 1.2S or 2S). If so I would like to know
>how good the analysis obtianed and if any problems were encountered, and of
>any recent literature of this matter. 
>                                Thanks 
>                                Hilal A.  Lashuel)

Regarding the use of g(s*)t for low molecular weight proteins:

The dcdt method produces a peak of g(s*) vs s* whose maximum position
appears at a value of s* that is slightly less than the true value of s.
The amount by which it underestimates the true value is related to the
molecular weight of the protein and is essentially independent of the 
true value of s. 

The shift is 1% (0.99) at 60kDa, 2% (0.98) at 35kDa, 3% (0.97) at 25kDa, 4%
(0.96) at 18kDa. Below about 15-17kDa is it nearly impossible to get the
boundary to clear the meniscus and so it is recommended that the time
derivative method not be used below that range. This shift in g(s*)t,
arises from the way in which g(s*) is computed in that it does not fully
account for the effects of diffusion on the boundary shape. There is an
exact correction for this effect that I will publish soon BUT it nearly
completely obviates the automatic baseline subtraction that makes dcdt so
useful. 

This shift seems a small inconvenience, unless very accurate s values are
needed, for example, for shape analysis. In that case one could measure s
the "old fashioned way" using a synthetic boundary cell, using the
transport method or using second moment calculations. However, given the
uncertainties in the estimates of hyrdation factors, one might not find the
use of the correction factors objectionable. 

The above set of correction factors will do well in most "everyday"
situations. However, I strongly recommend that if you are interested in
getting more accurate S, D and M values from sedimentation velocity
analysis for proteins in the molecular weight range below 20K that you use
one of the direct fitting methdos like John Philo's Svedberg program or
Borries Demeler's finite element curve fitting program. Those methods work
very well as long as there is no significant concentration dependence. 


Walter Stafford
stafford@bbri.harvard.edu