From: John Philo <jphilo@mailway.com>
  To  : Bethell, Sue S <ssb25133@glaxowellcome.co.uk>
  Date: Wed, 24 Nov 1999 08:57:53 -0800

RE: Another what is going on?

Sue,

First, regarding the 'slow' sedimentation, it seems to me you are running at
far too low a rotor speed. For something in that size range I would normally
go to 60K, even without the glycerol and low temperature (which together
will increase the viscosity and therefore sedimentation time 2-fold versus
water at 20 C).

Second, regarding the precipitation/denaturation, what you are describing is
on the extreme end of the spectrum, but many proteins are sensitive to shear
stress and/or denaturation at air/water interfaces or on solid surfaces.

It is often overlooked that cosolutes such as sucrose which are normally
stabilizing (in that they increase the relative stability of the native
state) can actually aggravate surface-induced denaturation by driving the
protein to the surfaces. According to my partner Tsutomu Arakawa, an expert
in this area, glycerol is generally fairly neutral in that regard, but for
some proteins it does drive them to surfaces.

If this is a protein that has been refolded, a strong tendency to aggregate
with time or mild stress could also indicate that it is not properly folded.
Such a protein will also have a very low sedimentation coefficient for its
mass (high frictional coefficient ratio f/fo).

'Hope this helps,

John Philo, Alliance Protein Laboratories
www.ap-lab.com

-----Original Message-----
From: Bethell, Sue S [ssb25133@glaxowellcome.co.uk]">mailto:ssb25133@glaxowellcome.co.uk]
Sent: Wednesday, November 24, 1999 7:50 AM
To: rasmb@alpha.bbri.org
Subject: Another what is going on?


Dear all,

Have any of you had any experience with proteins that precipitate when they
are agitated?  I don't mean the good old reliable 'bubble test' (if the
solution froths there is protein in there).

This protein precipitates out when it is left GENTLY rocking or stirring
even after a couple of hours.  Dialysis has to be done over extended periods
without stirring.  It also does not like freeze-thawing, as it precipitates
upon thawing even in the presence of 30% glycerol.

I am running a velocity sedimentation experiment at the moment:  the MW of
the protein by sequence and mass spec is 37KD.  I am running it at 40000rpm
in 10% glycerol 4C.  Concentrations in absorbance units at 280nm are 1.2,
0.7 and 0.4AU.  The protein has just about cleared the meniscus after 4.5
hours.  I know that glycerol slows things down due to viscosity and all but
this really slow.

Anybody know what may cause a protein to precipitate when shaken or stirred
and why it may be so slow to sediment?   Shape?

Thanks in advance for any suggestions!!!!

Sue

Sue Bethell
Protein Technologies
Protein Science Unit
GlaxoWellcome R&D
Gunnelswood Road
Stevenage
Herts. SG1 2NY
UK

email: ssb25133@GlaxoWellcome.co.uk
tel:  (44) (0)1438-763419
fax: (44) (0)1438-764865