[RASMB] concentration range for non-ideal behavior

[Allen Minton]

John -

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.  .

Allen Minton

At 03:02 PM 1/22/2015, John Sumida wrote:
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>Dear RASMB,
>
>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.
>
>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.Â
>
>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?Â
>
>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.
>
>Once again thank you all for your thoughts and suggestions.
>
>Best regards,
>John Sumida
>University of Washington
>
>From: RASMB [mailto:rasmb-bounces at list.rasmb.org] On Behalf Of David Hayes
>Sent: Wednesday, January 21, 2015 6:25 PM
>To: RASMB
>Subject: Re: [RASMB] concentration range for non-ideal behavior
>
>Hi John,
>
>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.
>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.
>
>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.
>
>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.
>
>David
>
>
>
>
>From: John Sumida 
><<mailto:jpsumida at u.washington.edu>jpsumida at u.washington.edu>
>To: <mailto:rasmb at rasmb.org>rasmb at rasmb.org
>Sent: Tuesday, January 20, 2015 9:00 PM
>Subject: [RASMB] concentration range for non-ideal behavior
>
>Dear RASMB,
>
>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.
>
>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.
>
>Best regards
>John Sumida
>University of Washington
>
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