Hello,
we are working with a specific peptide and want to check whether it is stable in particular formulation or not. Is there a change in PI of peptides when there conformation is disturbed and could it be possible to do IEF to find out its stability?
Hi Heena,
I don't think peptide confirmation will influence its pI (maybe aggregation could..), but I do think that IEF can be stability-indicating: if you're peptide contains amino acids that can be modified upon storage (in formulation), such as asparagine/glutamine that can be deamidated, methionine/cysteine that can be oxidized, these modifications can alter the pI of the peptide.
In addition to the ones mentioned above, lysine glycation, arginine deimidation and/or N/C-terminal reactions, such as pyroglutamate formation can also influence the pI of a peptide (or protein).
Hope this helps!
Hi Heena,, what do mean by "we are working with a specific peptide and want to check whether it is stable in particular formulation or not"
Are you talking about precipitation/conformation problems?
Prepared 10-30mer peptides do not regularly degrade unless there is proteinase contamination.
Hello Stefansson,
Actually the peptide is calcitonin and we want to check whether it is stable in formulation we are using , it is not precipitating in it but cant say if have some conformation changes. It is 32 amino acid peptide . Can we check it with PI changes?
Hi Heena
If you are focusing on secondary structure - Try to do circular dichroism (CD). It will give you exact idea of the conformation of your poly peptide. Hope that it possess a secondary structure.
If PI changes are your concern, then u need to titrate and calculate for each formulation. Changes in PI may not be substantial as your peptide is very small.
U can consult IMTECH / IISER for CD, if the facility is not in NIPER.
Dr. P. Guptasarma may be glad to help u at IISER.
All the Best
I support Arshad's suggestion, in addition you could try IR-spectroscopy (amide bands) to check for secondary structure changes. For aggregation laser light scattering is fastest, size exclusin chromatography is an alternative.
As to pI, computer programs calculate it from the pKa values of amino acids in water, by solving the Henderson-Hasselbalch-equation (see http://isoelectric.ovh.org/ for further discussion). However, these pKa-values are not applicable inside a protein. For example, there is considerable energy penalty for dissociation in the hydrophobic core of a protein or if the resulting charge is the same as one nearby. See
@ARTICLE{Gri-09,
title = {A summary of the measured p{K} values of the ionizable groups in folded proteins},
author = {G.R. Grimsley and J.M. Scholtz and C.N. Pace},
journal = {Protein Sci. },
year = {2009},
pages = {247-251},
volume = {18},
abstract = {pKa Werte von Aminos{\"a}uren in Proteinen im Durchschnitt gleich denen in kurzen Peptiden, aber mit extremen Ausrei{\ss}ern},
doi = {10.1002/pro.19},
language = {engl},
}
@ARTICLE{Win-04b,
title = {Determination of the net charge (valence) of a protein: a fundamental but elusive parameter},
author = {D.J. Winzor},
journal = {Anal. Biochem.},
year = {2004},
pages = {1-20},
volume = {325},
abstract = {Review: Bestimmung der Ladung in Abh{\"a}ngigkeit vom pH},
doi = {10.1016/j.ab.2003.09.035},
language = {engl},
}
Due technical reasons the web server for pI has been moved from http://isoelectric.ovh.org to http://isoelectric.org
Details about Isoelectric Point Calculator and comparison to all other pI algorithms (>15 methods) in Article IPC – Isoelectric Point Calculator
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