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From: Jacob Lebowitz <lebowitz@helix.nih.gov>
To : Brian M. Baker <baker@xtal200.harvard.edu>
Date: Thu, 17 May 2001 13:51:31 -0400
Re: hydrogen exchange with D2O
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Brian,
I don't believe that it is possible to combine the Reynolds and Tanford
approach of density matching of a single detergent with Edelstein &
Schachman procedure. The latter uses the solution of simultaneous
equations from two sedimentation equilibrium experiments performed at
different densities, e.g. H2O and D2O solutions, to solve for Mw and
vbar. If the density does not match the detergent then you will have a
contribution from M(1-vbarp) of the bound detergent. Hence, you can not
set up simultaneous equations. Jo Butler's approach, based on Eisenberg's
work, is quite rigorous but does require 10 mg of protein which we often
do not have.
I suggest you check out the following papers with respect to the
determination of molecular weights of glycosylated proteins.
Lewis MS, Junghans RP
Ultracentrifugal analysis of molecular mass of glycoproteins of unknown or
ill-defined carbohydrate composition.
Methods Enzymol. 2000;321:136-49.
Shire, SJ (1994) in Modern Analytical Ultracentrfiguation (Schuster, T.M.
and Laue, T.M.eds.) pp.261-297, Birkhauser, Boston
Fairman et al. Analytical Biochemistry 270, 286-295 (1999). This work
includes mass spectral analysis and is the most rigorous analysis.
For detergent solubilized membrane proteins check out.
Schubert, D. and Schuck, P. Progr.Colloid. Polym. Sci. 86, 12-22 (1991)
You should also obtain additional references from Schubert's lab since he
has extensively focused on detergent solubilized
membrane proteins. I believe that Schubert's lab used two different
detergents and did density matching and then solved for M and vbar for the
glycoprotein, i.e. a version of Edelstein-Schachman. Sorry, I don't have
the reference. In my experience the E-D approach has not worked too well.
I would focus on the approach described by the first 3 references coupled
with the references re the use of detergents. Density matching need not be
only with D2O. Sucrose and glycerol were used by Schubert's lab, Mayer et
al. 1999. Prog.Colloid Polym. Sci 113,176-181. We have successfully used
sucrose with a membrane solubilized viral protein complex.
Good luck with your analysis.
Jack Lebowitz
At 07:28 PM 5/15/01 -0400, you wrote:
>Greetings all,
>I'm planning on doing the Reynolds and Tanford (PNAS 73, 4467) approach with
>a detergent solubilized membrane protein to get at the protein molecular
>weight without the detergent contribution (protein is supposed to be a
>stable trimer and it does not seem to like C8E5). One thing I'm unsure about
>is correcting for hydrogen/deuterium exchange when using D2O (yes, I gave up
>on getting D2O18). In plots of apparent M(1-vbar*rho) vs. solution density,
>I've seen it mentioned that corrections are applied for exchange, but I'm
>not sure how this is done. Can anybody steer me in the right direction?
>
>I know how to correct for exchange when doing the Edelstein & Schachman
>procedure for getting the vbar & MW of the protein/detergent complex - I
>think I'm missing an obvious link between the two approaches.
>
>Thanks much,
>-Brian
>
>============================================
>Brian M. Baker
>Department of Molecular and Cellular Biology
>Harvard University
>baker@crystal.harvard.edu
>(617) 496-6074
>http://baker.mcb.harvard.edu
Jacob Lebowitz, PhD
Molecular Interactions Resource
Division of Bioengineering and Physical Science, ORS
National Institutes of Health
Mail: Bldg. 13 Rm. 3N17
Office Bldg. 13 Rm. 3E49
13 South Drive
Bethesda, MD 20892 - 5766
Tel: (301) 435-1955
Fax: (301) 480-1242
email: lebowitz@helix.nih.gov
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Content-Type: text/html; charset="us-ascii"
<html>
Brian,<br>
<br>
I don't believe that it is possible to combine the Reynolds and Tanford
approach of density matching of a single detergent with Edelstein &
Schachman procedure. The latter uses the solution of simultaneous
equations from two sedimentation equilibrium experiments performed at
different densities, e.g. H2O and D2O solutions, to solve for Mw and
vbar. If the density does not match the detergent then you will
have a contribution from M(1-vbarp) of the bound detergent.
Hence, you can not set up simultaneous equations. Jo Butler's
approach, based on Eisenberg's work, is quite rigorous but does
require 10 mg of protein which we often do not have. <br>
<x-tab> </x-tab><font face="Times New Roman, Times" size=4>I
suggest you check out the following papers with respect to the
determination of molecular weights of glycosylated proteins.<br>
</font> <font color="#0000FF"><u>Lewis MS, Junghans RP<br>
</font></u>Ultracentrifugal analysis of molecular mass of glycoproteins
of unknown or ill-defined carbohydrate composition.<br>
Methods Enzymol. 2000;321:136-49. <br>
<br>
Shire, SJ (1994) in Modern Analytical Ultracentrfiguation (Schuster, T.M.
and Laue, T.M.eds.) pp.261-297, Birkhauser, Boston<br>
<br>
Fairman et al. Analytical Biochemistry 270, 286-295 (1999). This work
includes mass spectral analysis and is the most rigorous analysis.<br>
<br>
For detergent solubilized membrane proteins check out.<br>
<br>
Schubert, D. and Schuck, P. Progr.Colloid. Polym. Sci. 86, 12-22
(1991)<br>
<br>
You should also obtain additional references from Schubert's lab since he
has extensively focused on detergent solubilized<br>
membrane proteins. I believe that Schubert's lab used two different
detergents and did density matching and then solved for M and vbar for
the glycoprotein, i.e. a version of Edelstein-Schachman. Sorry, I don't
have the reference. In my experience the E-D approach has not
worked too well. I would focus on the approach described by the first 3
references coupled with the references re the use of detergents. Density
matching need not be only with D2O. Sucrose and glycerol were used by
Schubert's lab, Mayer et al. 1999. Prog.Colloid Polym. Sci 113,176-181.
We have successfully used sucrose with a membrane solubilized viral
protein complex. <br>
<br>
Good luck with your analysis.<br>
<br>
Jack Lebowitz<br>
<br>
<br>
<br>
<br>
<br>
<br>
<br>
<br>
At 07:28 PM 5/15/01 -0400, you wrote:<br>
<blockquote type=cite cite>Greetings all,<br>
I'm planning on doing the Reynolds and Tanford (PNAS 73, 4467) approach
with<br>
a detergent solubilized membrane protein to get at the protein
molecular<br>
weight without the detergent contribution (protein is supposed to be
a<br>
stable trimer and it does not seem to like C8E5). One thing I'm unsure
about<br>
is correcting for hydrogen/deuterium exchange when using D2O (yes, I gave
up<br>
on getting D2O18). In plots of apparent M(1-vbar*rho) vs. solution
density,<br>
I've seen it mentioned that corrections are applied for exchange, but
I'm<br>
not sure how this is done. Can anybody steer me in the right
direction?<br>
<br>
I know how to correct for exchange when doing the Edelstein &
Schachman<br>
procedure for getting the vbar & MW of the protein/detergent complex
- I<br>
think I'm missing an obvious link between the two approaches.<br>
<br>
Thanks much,<br>
-Brian<br>
<br>
============================================<br>
Brian M. Baker<br>
Department of Molecular and Cellular Biology<br>
Harvard University<br>
baker@crystal.harvard.edu<br>
(617) 496-6074<br>
<a href="http://baker.mcb.harvard.edu/" eudora="autourl">http://baker.mcb.harvard.edu</a></blockquote><br>
<div>Jacob Lebowitz, PhD </div>
<div>Molecular Interactions Resource </div>
<div>Division of Bioengineering and Physical Science, ORS </div>
<div>National Institutes of Health </div>
<div>Mail: Bldg. 13 Rm. 3N17</div>
<div>Office Bldg. 13 Rm. 3E49</div>
<div>13 South Drive </div>
<div>Bethesda, MD 20892 - 5766 </div>
<div>Tel: (301) 435-1955 </div>
<div>Fax: (301) 480-1242</div>
<div>email: lebowitz@helix.nih.gov</div>
</html>
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