Measuring peptide concentration

Emir Khatipov khatipovNO at NOuchicago.edu
Tue Mar 19 22:08:27 EST 2002


Artem,
If you can look it up quickly, could you specify how different is absorption
of 2 cycteines bonded by disulfide compared to the same 2 Cys reduced? Say
if I have a 25-mer peptide without aromatic residues but with 2 cysteines
separated by e.g. 5 residues, would that be possible to quantitatively
determine spectrophotometrically the ratio of oxidized (cyclized) to reduced
(linear) species in solution (water)? I know about DTNB assay, but want to
have a second method for comparison. I am sure I can find everything in the
books, but if you have any experience you could share, I'd much appreciate
your input.
Thanks,
Emir

"Artem Evdokimov" <AEVDOKIMOZ at cinci.rr.com> wrote in message
news:1JQl8.171398$Hu6.39493731 at typhoon.neo.rr.com...
> Yes, Cys has an absorption peak there. Depending on the state - CyS-SyC is
> different from two Cys !
>
> No worries :)
>
> A.G.E.
>
> "Emir Khatipov" <khatipovNO at NOuchicago.edu> wrote in message
> news:xjOl8.347$s4.26393 at news.uchicago.edu...
> > So these are cysteines that give some fluorescence to my non-aromatic
> > peptides! It's never too late to learn. Thanks for opening my eyes a
> little
> > bit wider, Artem :-)
> > Cheers,
> > Emir
> >
> > "Artem Evdokimov" <AEVDOKIMOZ at cinci.rr.com> wrote in message
> > news:GBxl8.166129$Hu6.38723197 at typhoon.neo.rr.com...
> > > I disagree on one point : Expasy server does NOT calculate extinciton
if
> > you
> > > don't have certain types of AA's in your sequence.
> > > For example 'AAAAAA' results in
> > >
> > > -----------
> > > Extinction coefficients:
> > >
> > > As there are no Trp, Tyr or Cys in the region considered, your protein
> > > should not be visible by UV spectrophotometry.
> > > -----------
> > >
> > > There's a reason for it - while some absorption at 280 will indeed be
> > > present, it won't be characteristic, and it would be overwhelmed by
> > > non-specific absorption of crud.
> > >
> > > Otherwise, way to go Emir :)
> > >
> > > A.G.E.
> > > "Emir Khatipov" <khatipovNO at NOuchicago.edu> wrote in message
> > > news:pyul8.311$s4.23478 at news.uchicago.edu...
> > > > Use Protparam tool at www.expasy.ch/tools to calculate molar
> extinction
> > > > coefficient of your peptide and use it for your spectrophotometric
> > > > measurements. Peptides absorb light at 280nm even if they don't
> contain
> > > any
> > > > aromatic residues. I hope it is needless to say that you should use
> same
> > > 10%
> > > > DMSO in PBS as a reference. If you believe there are impurities in
> your
> > > > samples, I would suggest determining absorption spectra of your
> peptide
> > > > solutions at ~200-350nm (or at least 250-330nm). If your sample is
> pure,
> > > you
> > > > should see a nice peak at around 276-280nm. Be aware that peptide
bond
> > > > absorbs at 214nm, but there might be a lot of interference from
> > impurities
> > > > you may have in your sample.
> > > >
> > > > Emir
> > > >
> > > >
> > > > "JB" <j.bukczynskiDELETE_ME at utoronto.ca> wrote in message
> > > > news:b87a9ugslasbigp8lm4dhkqc4p0soe0se7 at 4ax.com...
> > > > > Hi,
> > > > >
> > > > > Does anyone know how to measure peptide concentration using a
> > > > > spectrophotometer? I have it dissolved in 10% DMSO in PBS. Any
help
> > > > > would be appreciated. Thanks,
> > > > >
> > > > > Jacob
> > > >
> > > >
> > >
> > >
> >
> >
>
>





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