From: Jo Butler <pjgb@mrc-lmb.cam.ac.uk>
  To  : baker@xtal200.harvard.edu
  Date: Wed, 16 May 2001 12:40:07 +0100

hydrogen exchange in D2O

Dear Brian,

While what you suggest is perfectly feasible, I would ask why you are not 
simply using the direct method and measuring the density increment, i.e. 
the value which you are actually calculating with (1-vbar*rho) ?  If you 
are familiar with Casassa and Eisenberg (1964; Adv. Prot. Chem. 19, 
287-395), you will know that measuring d(rho)/d(c2) directly will give you 
Mr for the species whose concentration you use for c2, i.e. if you measure 
the concentration of the protein alone (e.g. by amino acid analysis for an 
accurate value) you will get the mass of the protein without any 
contribution from the bound detergent.  Conveniently, the measurement of 
densities also gives you the correct value for the solvent density.
Because this is a direct measurement of the relevant parameter, it gives 
better values than the earlier, indirect methods, which rely on second 
order differences.  Densities are readily measured with the Paar 
oscillating densitometer (Kratky et al.; 1973; Met. In Enz. 27, 98-110). 
This requires <1 ml solution, that at as high a concentration as reasonable 
(to get a good density difference in d(rho)/d(c2)) and the only problem 
with the detergent is in getting it equilibrated between solution and 
solvent - for some detergents lengthy dialysis is fine, with others gel 
permeation chromatography is the best way, provided the protein/detergent 
micelles separate from the detergent alone micelles.  This is anyway a 
problem for the sedimentation studies and, since one recovers the density 
sample, it can be used for at least one of your centrifuge runs.
I have advocated using this method before, at one of the UK AUC Users Group 
meetings (Butler; 1998; Biochem. Soc. Trans. 26, 749-753) and use it quite 
frequently with various membrane proteins (e.g. Cabezon et al.; 2000; J. 
Biol. Chem. 275, 25460-25464).

Jo

P.J.G. Butler,
MRC Laboratory of Molecular Biology,
Hills Road, Cambridge, CB2 2QH, UK.
Tel. +44 (0)1223 402296