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John -<br><br>
Nonideal behavior is due to nonspecific interactions between solute
molecules that may be weak on a pairwise basis, but become significant
with increasing solute concentration. These interactions may be
either repulsive or attractive and depend upon a variety of factors,
including pH, ionic strength, and temperature. I am attaching a
paper with a very simplified analysis of the pH-dependent nonideal
behavior of bovine serum albumin. The bottom line is that there can
be no "general rule" for a concentration below which nonideal
behavior is negligible. Under some conditions a solution may behave
almost ideally up to quite high concentration, and under other conditions
nonideal behavior will kick in at low concentrations. You can see
this in Fig 1 of the attached article. Depending upon pH and the
concentration and type of counterions, a large polyanion like DNA can
behave nonideally at concentrations below 0.1 mg/ml. .<br><br>
Allen Minton<br><br>
At 03:02 PM 1/22/2015, John Sumida wrote:<br>
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Dear RASMB,<br>
<br>
David and all who have responded, thank you for your comments. I think in
the aggregate of comments received I have my answer and more
questions.<br>
<br>
In the range of concentrations measured 0.2 – 4.9mgs/ml the slope of the
experimental s with concentration is negative but only slightly so
(slope=-0.033 s/c, where c is the mass concentration g/cc). Â The
extrapolated s at 0mgs/ml is 6.88, given the information received in
these posts I think that at 4.2 mgs/ml I ought to expect a ~4.4% decrease
in s due solely to excluded volume effects. If so the expected
s-exp at 4.9mgs/ml ought to be 6.58. What is actually observed is a value
of 6.72s. So I think what David and others have suggested is
probably accurate, namely that self-association is offsetting the
non-ideality here. <br>
<br>
This raises some questions perhaps that would be good to test. I
can’t shut off self-association but perhaps one could slow it down by
reducing the temperature from 20C to 4C? I think excluded volume is only
weakly dependent on temperature and self-association more strongly
dependent on temperature? <br>
<br>
Interestingly, the sw vs conc relationship changes as a function
of buffer, whereas the slope of relationship is only weakly negative in
HEPES (0.01M HEPES, 150mM NaCl, pH7.5) in PBS (0.137M NaCl) it is
strongly positive.<br>
<br>
Once again thank you all for your thoughts and suggestions.<br>
<br>
Best regards,<br>
John Sumida<br>
University of Washington<br>
<br>
<b>From:</b> RASMB
[<a href="mailto:rasmb-bounces@list.rasmb.org" eudora="autourl">
mailto:rasmb-bounces@list.rasmb.org</a>] <b>On Behalf Of </b>David
Hayes<br>
<b>Sent:</b> Wednesday, January 21, 2015 6:25 PM<br>
<b>To:</b> RASMB<br>
<b>Subject:</b> Re: [RASMB] concentration range for non-ideal
behavior<br>
<br>
Hi John,<br>
<br>
The opposite of non-ideality is reality. All real molecules take up
space, so all real molecules sediment with some excluded volume
non-ideality. Charge is another source of non-ideality: the
ideal model of a molecule, besides being a geometric point with no
volume, also has no attractive or repulsive forces.<br>
The type of non-ideality that is not avoidable is excluded volume.
So it is not exactly mg/ml that determines whether or not the
non-ideality is significant, but the excluded volume. For most
proteins it is reasonable to see little effect from concentration alone
up to 1-2 mg/ml. But, put a molecule in a salt free buffer where
charge is not screened (Actin) or take a molecule that has a really big
carbohydrate attached (Pegasys) and nonideality can be evident at 0.5
mg/ml and below.<br>
<br>
In sedimentation velocity, the other non-ideality is the back flow.
When a real molecule moves down the cell, real buffer has to move up the
cell to fill in the otherwise to be empty spot where the molecule
was. This hydrodynamic non-ideality is also always there, but
usually negligible below 1-2 mg/ml.<br>
<br>
Did you do a concentration series of your antibody? If so, it is
expected that around 5 mg/ml; you would have measurable concentration
dependence of the sedimentation coefficient. If your peak moves the
same S at 0.5 and 5 mg/ml I would suspect self association is adding S
and offsetting the concentration dependence of S. Static light
scattering is helpful to use as an orthogonal method when self
association and non-ideality balance out in AUC-SV.<br>
<br>
David<br>
<br><br>
<br>
<div align="center"><br>
</div>
<b>From:</b> John Sumida
<<a href="mailto:jpsumida@u.washington.edu">
jpsumida@u.washington.edu</a>><br>
<b>To:</b> <a href="mailto:rasmb@rasmb.org">rasmb@rasmb.org</a> <br>
<b>Sent:</b> Tuesday, January 20, 2015 9:00 PM<br>
<b>Subject:</b> [RASMB] concentration range for non-ideal behavior<br>
<br>
Dear RASMB,<br>
<br>
My recollection is that concentrations in excess of 1.0 mg/ml should be
avoided due to the possibility of non-ideal interactions that would
complicate the analysis of SV and SE measurements. However in two
recent cases (in one case a mAb was being studied, and in another a
globular viral capsid protein), we exceeded this value by a factor of
five and failed to observe any evidence of non-ideal behavior. In both
cases, it was possible to fit both SV and SE data without assuming any
non-ideal interactions when the experiment was performed in PBS or
HEPES.<br>
<br>
I would be curious to hear if it is the experience of the community that
non-ideality is commonly to be expected at concentrations >1.0mg/ml or
whether the concentration cutoff for non-ideality is less of an actual
cutoff being more a function of the particular properties of the molecule
being studied.<br>
<br>
Best regards<br>
John Sumida<br>
University of Washington<br><br>
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