From: Arthur Rowe <Arthur.Rowe@nottingham.ac.uk>
  To  : kgf@mail.csb.yale.edu, rasmb@bbri.org
  Date: Fri, 25 Jun 1999 14:48:57 GMT0BST

re: small peptides - SE and non-ideality

Hi Karen, and all RASMBers !

Karen, you write -

"However, when I assume a "normal" vbar (~0.73) and calculate the peptide
MW from the reduced MW, I am finding that it is much smaller than I
expect. Since the ideal, single species model describes the data so
well, and since it is the simplest model, I conclude that nonideality is
probably not causing this depressed MW. (Any comments anyone??) "

Actually, testing for whether a given equilibrium has or has not got non-ideality 
present by doing a simple ideal fit is hazardous.  For purposes of illustration, let us 
take perfect, simulated SE data (LSE, c(b)/c(m) ~3, c(m) = 2 mg/ml), for 2BM = 40. i.e. 
only a modest solute concentration, and a degree of non-ideality which you might 
well observe if charge effects were present.  

Let us then add on simulated error of around +/- 1% at mid-point (SD constant, not 
in % terms, of course).  And then do a fit of the IDEAL1 type, floating the M value 
and the baseline.  

What do we get ?  For those who can down-load it, the graphs are attached. 
Basically, a very nice distribution of residuals is seen, no way you could infer 
anything about non-ideality, but an M-value of only 76% of the true value !  With a 
seriously non-zero baseline reported.  

Forcing the baseline to be zero (i.e. its true value in the simulation) M although still 
too low (88% of true) is pretty close to M/(1 + 2BMc).  

So - in such cases we cannot, I am afraid, "conclude that nonideality is probably not 
causing this depressed MW".  

Approaches to this problem include doing a Z-T difference equation plot (Rowe & 
Rowe, BBA, 222, 647-659;  FEBS Letters, 9, 124 (both 1970).  This works fine if the data 
are smooth enough, returning an M value at zero concentration, as well as an 
estimate for 2BM (symbol used : alpha).  And of course, multiple SE expts over a 
range of solute concentrations should help - but bear in mind (see above) that you 
must NOT float the baseline. 

All very best to you and to all

Arthur



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Professor Arthur J Rowe
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University of Nottingham
School of Biological Sciences
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