From: Joel Mackay <j.mackay@biochem.usyd.edu.au>
  To  : rasmb@bbri.org
  Date: Fri, 25 Jun 1999 08:58:33 +1000

re: small peptides

Dear Karen,
I have also found exactly the same situation with peptides of around 3 kDa.
The measurements were done in water alone (no salts or buffer) and the
residuals were very nice, with the mass coming out as around 1.5 kDa.
Repeating the measurements in PBS yielded the correct mass with residuals
that were just as good, so I presumed it was an effect related to the lack
of buffer, although I never knew exactly what. As you say, if the residuals
are good, I would have thought it couldn't be non-ideality, but I am
relatively new to this fieldl, so am not certain.
Hope this is of some help,
berst regards,
Joel Mackay 
>
>I am also in the midst of a project in which I am looking at the MW of a
>small, synthetic peptide. Just this week in fact I have obtained some
>useful equilibrium data which are best described (using NONLIN) by an
>ideal, single species model. The statistics for the fit are quite
>stellar for my machine (SRV=~4E-03 with ~900 degrees of freedom, 3
>speeds at one concentration). The residuals are beautifully small and
>random.
>
>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??) And
>actually, my first instinct is to question the value for the vbar. While
>I have never seriously looked into this issue in detail, I have the
>impression that the calculation of vbar from composition loses its
>precision as the "protein" sample becomes small enough to be considered
>a "peptide". Since I am not equipped with a precision densimeter, as a
>first approach towards addressing whether the peptide vbar is anomolous
>or not, I set up an H2O/D2O mixing experiment a la Edelstein and
>Schachman. I do not yet have the results from this experiment, but I
>would welcome any comments on this strategy.
>
>I should also mention that fussing with the vbar value in the
>calculations has been quite amusing, since I can vary my peptide MW by
>+/- 100%, depending on the value I choose. I will need to get up in the
>0.85 range in order for the MW to come out with reasonable agreement
>with the prediction. I'm thinking that this may not be so outrageous,
>since the synthetic peptide sample also contains some engineered,
>organic portion. I will probably also try to calculate a vbar from the
>atomic composition, taking into account the organic portion.
>
>Comments anyone??
>
>-Karen Fleming
>Yale University
>kgf@csb.yale.edu
> 
************************************************************************
Dr Joel Mackay			ph +61-2-9351-3906
ARC Research Fellow			fax +61-2-9351-4726
Department of Biochemistry
University of Sydney
NSW 2006 Australia
http://www.biochem.usyd.edu.au/~joel/
************************************************************************