From: Jim Sparrow <jsparrow@athero.med.bcm.tmc.edu>
Subject: RE: peptide oxidation?
Date: Fri, 24 Jan 97 14:38:00 C
To: Recipients of ABRF List <abrf@aecom.yu.edu>
Stephen,
I have observed a similar phenomenon with a 38 residue peptide which we
wished to cyclize. I believe that in our case we had a conformational
change that was reversed by the addition of DTT and which we could also
induce by a pH change. When we oxidized with I2 or Fe(CN)6 we got a later
eluting peak that had the correct mass which was reduced by DTT to the
earlier peak.
Hope this helps, Jim Sparrow
-------------------------------------------------------
Dr. James T. Sparrow
Dept. of Medicine
Baylor College of Medicine
One Baylor Plaza
Houston, TX 77030
Phone: 713 798-4156
FAX: 713 798-4121
email: jsparrow@bcm.tmc.edu
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From: abrf-request
To: Recipients of ABRF List
Subject: peptide oxidation?
Date: Thursday, January 23, 1997 5:09PM
We have synthesized a peptide 38 residues in length which was purified
by reverse phase HPLC to a single peak. The peptide has 2 cysteines and
2 tryptophans and no methionines. Further analytical methods outline
below indicate an oxidation event that
I am having trouble identifying. Here are my observations:
CE of freshly dissolved peptide indicates three peaks. One main peak at
70% and two minor peaks at about 15%. As the solution sits at room
temperature the biggest peak diminishes with time and eventually
disappears. One of the other peaks increases to eventually account for
all of the integrated area of the lost peak. This appears to be a
simple formation of disulfides (either inter or intra chain) or
oxidation.
Mass analysis indicates one major peak that corresponds to the
theoretical reduced form of the peptide and two minor peaks (-104 amu
and +146 amu). And there are no changes in the mass results with time
in solution. Therefore no TRP oxidation or cysteic acid.
Ellmans testing indicates 100 % free thiols for all time points in
solution. The ellmans tests were carefully controlled with standards of
free cysteine and reduced and cyclized standard peptides. The 100 %
result was +/- 1% over three experiments.
Amino acid analysis of an oxidized peptide indicated 100% cysteic acid.
We repeated the CE analysis versus time of dissolution at a higher pH
(9.0) and found the same results but somewhat slower conversion from one
peak to another. Next we added DTT to the solution and this converts
the CE pattern back to the freshly diluted peptide.
Am I missing something obvious here? If we had disulfide formation, the
ellmans tests would have lower free thiols with time after disolution.
In addition, the AAA cysteic acid quantity would be reduced (oops,
lowered). If there were interchain disulfides we would see a change a
doubling of the mass. If there was oxidation of TRP or CYS we would
have seen a change in the mass. Yet there appears to be a DTT reversible
oxidation (?).
Does anyone have any suggestions?
Stephen Kaminsky
Department of Peptide Chemistry
United Biomedical, Inc.
25 Davids Drive
Hauppauge, New York 11788
(516) 273-2828
(516) 273-1717 fax
E-mail: stevenk@cerfnet.com