[RASMB] High sucrose concentrations in sed velocityexperiments

[John Correia]

vanHolde & Baldwin (JPhysChem 62, 1958 paper) measured D for sucrose from Archibald or approach to equilibrium experiment.  Got 4.93 to 5.00 (10-6) cm2sec-1 at 25oC which agreed with Gosting diffusion measurement - using the MW you can calculate S - they also report Gosting values for sucrose (vbar = 0.618 ml/gm).

>>> "John Philo" <jphilo at mailway.com> 11/14/05 11:01 AM >>>
David,

Yes but in order to use this you must know the sedimentation coefficient of
the small molecule and have all the necessary density and viscosity data. 

In that regard, does anyone actually have a good sedimentation coefficient
for sucrose, trehalose, and/or mannitol? 

John

-----Original Message-----
From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On
Behalf Of David Scott
Sent: Monday, November 14, 2005 7:39 AM
To: Matthew.Parker at immunogen.com; rasmb at rasmb-email.bbri.org
Subject: Re: [RASMB] High sucrose concentrations in sed velocityexperiments


Hello Matt,

Basically, small solutes will form a dynamic density gradient when you
perform sedimentation velocity. If there are enough around, then you have to
correct for it, which appears to be what you have here. We have a similar
problem as we work with extreme halophilic proteins that are happy only in
molar salt concentrations. Luckily Peter Schuck has written a nice routine
in SEDFIT to correct for this (under options, select inhomogenous gradient,
click "no" to compressible solvent and "yes" to dynamic gradient...). It
takes longer for everything to fit, but gets there in the end. 

The full and gory details are in a Biophys. Chem. paper:

P. Schuck (2003) A model for sedimentation in inhomogeneous media. I.
Dynamic density gradients from sedimenting co-solutes.  Biophysical
Chemistry 108:187-200

and further details of its implementation in SEDFIT are on Peter's website
under:

http://www.analyticalultracentrifugation.com/dynamic_density_gradients.htm


Have fun!

Dave Scott.





Dr. David J. Scott
Lecturer in Physical Biochemistry
National Centre for Macromolecular Hydrodynamics
School of Biosciences
University of Nottingham
Sutton Bonington
Leicestershire
LE12 5RD
Phone: +44 (0) 115 951 6221
Fax: +44 (0) 115 951 6142
Email: dj.scott at nottingham.ac.uk
http://www.nottingham.ac.uk/biosciences/foodsci/academic/scott.html 

>>> "Parker, Matthew" <Matthew.Parker at immunogen.com> 11/14/05 3:24 pm 
>>> >>>
            Hi. We recently ran sedimentation velocity on some samples that
contained 10% sucrose in the buffer. We obtained results for the c(s)
distribution that didn't agree well with what we saw by SEC, and the
s-values were also shifted to significantly lower values than what we would
expect (and that we have seen in other formulations) for an IgG. It was
suggested to us that our sucrose concentration was high enough to create a
sucrose gradient in the cell during the course of the run, and that this
could cause the relative peak areas in the c(s) plot to be inaccurate.



            Does anybody have any comments about this, or suggestions about
how high in sucrose content you can go before this starts to become a
problem?



            Thanks,



            Matt



Matthew Parker, Ph.D.

Analytical and Pharmaceutical Sciences

Immunogen, Inc.

Cambridge, MA



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