From: Jo Butler <pjgb@mrc-lmb.cam.ac.uk>
  To  : noland@quorum.tamu.edu
  Date: Tue, 1 Sep 98 12:04:48 +0000

Re: Measuring Association Constants in 2 M Urea

>Date: Mon, 31 Aug 1998 14:23:34 -0500
>From: Brian Noland <noland@quorum.tamu.edu>
>To: rasmb@bbri.harvard.edu
>Subject: Measuring Association Constants in 2 M Urea
>
>To whom it may concern,
>I would like to measure an association constant for the alpha subunit of
>bacterial luciferase in 2 M urea.  In buffer, the alpha subunit is a
>weakly associating system with a dissociation constant in the low
>micromolar range.  This protein has an equilibrium folding intermediate
>which is highly populated in 2 M urea.  I am guessing that the change in
>the structure will change the association constant.  For a sedimentation
>equilibrium experiment I don't know if there is a way to calculate the
>partial specific volume of the protein in 2 M urea.  Also, are there ways
>to get good association constants using sedimentation velocity
>experiments?  I would appreciate any advice from those who have ideas on
>this matter.
>
>Thank you,
>Brian Noland
>
>Brian Noland
>Dept. of Biochemistry & Biophysics
>Texas A&M University
>College Station, TX 77843
>noland@quorum.tamu.edu
>(409) 845-8971
>

Dear Brian,

The correct answer is to measure the density increment and solvent
densities and
then calculate the apparent partial specific volume, which will then allow
for
any binding of either urea or water.  While calculation can get one so
far, it
can never allow for unknown binding.  If you use the apparent partial
specific
volume at dialysis equilibrium, you get the molecular mass of the
component
whose concentration is used for measuring the density increment (i.e. if
protein
alone is measured, by amino acid analysis, the mass is that of the
protein):
Casassa, E. F. & Eisenberg, H. (1964). Thermodynamic analysis of
multicomponent
solutions. Adv. Prot. Chem. 19, 287-395.

I you want to get an idea of the partial specific volumes measured in
urea
solutions, we published them at various concentrations (1.3, 1.7, 3.0 and
5.0 M)
for the HPV-16 E2 DNA-binding domain: Mok, Y.-K., de Prat Gay, G., Butler,
P. J.
& Bycroft, M. (1996). Equilibrium dissociation and unfolding of the
dimeric
human papillomavirus strain-16 E2 DNA-binding domain. Prot. Sci. 5, 310-319.

I hope that these remarks are helpful,
Yours sincerely,


-----------------------------
Jo Butler, 
MRC LMB,
Cambridge, UK.
Tel:+44 1223 248011
FAX:+44 1223 213556
Email: pjgb@mrc-lmb.cam.ac.uk
-----------------------------