From: H. Olin Spivey <ospivey@bmb-fs1.biochem.okstate.edu>
  To  : "Bethell, ">Sue S" , rasmb@alpha.bbri.org
  Date: Wed, 24 Nov 1999 11:21:13 -0600

Re: Another what is going on?

Dear Sue,

   Yes, I have encountered several such proteins.  Indeed I am very skilled
at denaturing proteins, but this is well documented by others as well.
Some examples are rabbit muscle glyceraldehyde-3-P dehydrogenase (GAPD) in
the apo form, i.e, free of NAD(H), bovine glutamate dehydrogenase (GDH) in
Tris buffer, and avian lactate dehydrogenase (LDH).  There are a variety of
mechanisms that are likely responsible.  There is compelling evidence from
a large array of enzymes that they can exist in metastable states on the
way to denaturing.  In such states, it takes little agitation (increased
oxygenation and shear forces) to denature the enzyme.  The avian LDH is
very susceptible to denaturation by exposure to air - pellets during
purification must be kept covered with buffer.  The apoGAPD cannot be
stirred even gently without some denaturation at each stirring - a
phenomenon well documented by fluorescence titrations with ligands.  I
presume such metastability is true of numerous proteins that are not
enzymes, though my experience is limited to enzymes.

   There have been many articles in the past 5 years on novel protectants
against protein denaturation.  Having said this, I am amazed that I don't
find them in my database just now, but expect that I could locate them if I
searched longer.  There is a new technical term for these that I have
forgotten - perhaps these references could be rapidly located if someone
will remind us of this technical term.

   Freeze-thawing denatures many enzymes.  Sometimes this is due to poor
choice of the buffer and can be avoided with a better choice (Hill and
Buckley, 1991, Anal. Biochem. 192, 358-361).  Phosphate buffer is bad in
this regard as there is preferential distribution of the conjugate acid and
base between the ice and water phases during the freezing and thawing
(large pH changes during the phase changes).  Some proteins are cold labile
without freezing (e.g. pyruvate caroxylase) due to aggregation at low
temperatures (proteins with relatively large hydrophobic surfaces).

   The viscosity of your glycerol solution is about twice that of water at
20 deg. C according to the Sedenterp database , so your sedimentation time
is going to be twice as slow.  40,000 rpm is far too low for sedimentaion
velocity of a small protein like yours, even at room temp. with viscosity
of 1 centipoise.  Use 60,000 rpm.  If you have a synthetic boundary cell,
you can layer  buffer onto the sample at about 10,000 rpm and get a sharp
protein boundary without waiting for clearance of the meniscus.  Still
another method would be to use the improved Archibald method of Peter
Schuck (1998, Anal. Biochem. 259, 48-53), but this won't be as accurate.

   Good luck
   Olin
_____________________________________________________________



At 3:50 PM +0000 11/24/99, Bethell, Sue S wrote:
>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


H. Olin Spivey                       Phone: (405) 744-6192
Dept. Biochem. & Molec. Biology      Fax:   (405) 744-7799
246 NRC                              Email: OSpivey@Biochem.Okstate.Edu
Oklahoma State University
Stillwater, OK 74078-3035