I have been spending a good deal of deal trying to get an universal
procedure,and I
found out that no matter which method I tried,it does not work all the
time.
If the method is relatively small,and I will not be pinned down on size,a
reverse phase HPLC separation will do the job.If you can get same
column,conditions,etc.as the initial
run,it should work when combined with amino acid analysis.Once,I got a
peptide which gave
baseline resolution of two peaks of identical size.I found out that the
researcher
synthesized the derivatives,using the D,L-amino acid.
To prove the amount of D-amino acid is not so easy.I have seen
publications,and I tried
the procedures.They work for only a few amino acids.Probably,the best
method is that of
N.Nimura and T.Kinoshita,J.Chromat.,vol.352,169-177(1986).It relies on the
reaction of
primary amino acids with 0-phtaldehyde(OPA),in presence of sulfur compound
which is usually
the L-form.A ternary compound is formed,and it is possible to separate a
complex with
L-amino acid from D-amino acid,by the fact that there is a L-L and D-L
form.The authors and
Ihave been using N-acetyl-L-cysteine,although other sulfur compounds have
been used.
It is a precolumn derivatization and detection is fluorometric.I do not
think that any
other physico-chemical method will work.
Jim Bloom <Jim.Bloom.B@bayer.com>@aecom.yu.edu> on 07/26/2000 04:09:47 PM
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cc:
Subject: Racemization/polarimeters
Holding to the axiom that the older I get the less I find out that I
know...I have a peptide that I would like to keep "fresh", ie. I don't want
it
to "degrade" upon storage. Looking up ways that a peptide can degrade I
discovered that peptides that contain Asp residues can undergo racemization
(my
peptide just so happens to contain Asp). Looking in various old musty
organic
chem and biochemistry books I think I understand what racemization is. Now
the
question is...what analytical technique do I use to determine if my peptide
is
undergoing racemization? In theory, I would like to say it is 5% racemized
after X period of time and 15% racemized after Y period of time. I would
guess
that I need to buy something called a polarimeter and it will give me a
number
and I watch that number change over time. Can anyone help me out here? If
a
polarimeter will indeed do the job does one use a single wavelength machine
or
multiple wavelength? I have discovered three vendors: JASCO, Perkin Elmer
and
Ruldolph Instruments, Inc. Any vendor preference? Do I need special
cuvettes
or temp control or anything?
Weather Inconsequential: no mosquitoes, no thunderstorms that knock out
the
electricity just when you need the sump pump to keep the basement dry and
no
tornadoes. Just the occasional little ground tremor. No gophers/moles to
report this season.
Jim Bloom
Bayer Corp
Berkeley, CA
jim.bloom.b@bayer.com
510-705-7760
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