Radhika,
if you just mean resolution of conformational isomers on a small
preparative scale, this is possible, provided you perform it at a pH
value close to neutrality (where both acidic and basic catalyses are
negligible thus slowering the interconversion between different
conformations of the peptide chain) and of course on a refrigerated
column, also collecting fractions in ice-cooled vials. We also
achieved this in our lab on a proline-rich 13-peptide (see J
Chromatogr 1985, vol349, 117-130).
However, as the temperature raises back to ambient, equilibration will
take place again, more or less quickly depending on pH as well as on
the specific peptide sequence, always resulting in a mixture of
isomers. I would suggest to try to check your peptides, incubated
under "blank" conditions (the same used for the biological assay), by
RP-HPLC analysis with an almost neutral buffer, such as 50-100mM
ammonium acetate. Maybe in this way you can at least have an idea of
the isomer ratio in the conformational mixture that is actually being
assayed, and a time-course study might perhaps help to evaluate the
results. Regarding the identification of the isomers, this probably
requires further investigation of the peptides by NMR and CD.
Luisa
----------------------------------------------------------------------
Luisa Rusconi
Dept. of Biology
Biochemistry Lab/Protein Chemistry Core Unit
Pharmacia & Upjohn
Viale Pasteur, 10
20014 Nervaino (MI)
Italy
phone ++39-02-48383801
fax ++39-02-48383750
e-mail luisa.rusconi@eu.pnu.com
______________________________ Reply Separator _________________________________
Subject: PepSyn:Re:Peculiar.., more questions
Author: "Radhika Krishnan"<rkrishnan@smtplink.Coh.ORG> at Internet-europe
Date: 21/01/2000 9:11 AM
Thanks to everybody who resolved this problem for me.
I appreciate all the input.
I do have one reulting question however, these peptides will be used in a
biological assay done at room temperature. Will this affect the formation of one
isomer versus the other. I would assume that the trans is more stable than the
cis at higher temperatures, and vice versa. Is it at all possible to separate
these kind of isomers?
Thanks a lot.
Radhika.
City of Hope, Duarte.
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