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Subject: RE: PepSyn, Purification yields degradation products
Date: Tue, 15 May 2001 09:30:12 -0400
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Thomas,
You guessed it: almost definitely air oxidation to form disulfide linked
peptide dimers. If you like, you can confirm this via AAA or MS. There are
a lot of things you can do to keep your peptide from oxidizing in air,
mostly some basic precautions and also any of a variety of adulterants.
Precautions. Immediately upon pooling your prep fractions, degas them and
freeze them. Then process for lyophilization ASAP. When you make up the
purified sample for reinjections analysis, first degas the solvent. Then
overlay the sample with argon. Blanket your dry peptide with argon before
storing. Degas and blanket any time you make up a solution.
Adulterants. Add about 0.01-0.1% of a mild reducing agent to your solution.
Depending on what you can stand in your final peptide prep, that may include
thiols, phosphines, or other things. Dithiothreitol (DTT) is a popular
thiol, a cheap dithiol that dissolves easily in aqueous solvent mixtures.
2-mercaptoethanol (2ME) and 2-mercaptoacetic acid (2MA) are also popular
thiols. They smell pretty funky, but that's because they're volatile. That
means that if you only use around 0.01% thiol in your solvent, it will
theoretically all go away upon lyophilization. Pretty nifty, but not really
quantitative unless you lyophilize a few times in succession.
Tri-butylphosphine (Bu3P) is an excellent choice if you can't have competing
thiols in your peptide prep, e.g., because you want to react the peptide
through the cysteine sulfhydryl. Bu3P is poorly soluble in water, so make
up a stock solution of 1-10% in acetonitrile. Bu3P is also somewhat stinky
and volatile. As for other things, I don't personally recommend ascorbic
acid or ammonium iodide, although you may find others who will.
You also need to remember that pH is very important. Air oxidation of
thiols requires the thiolate anion species, and the pKa of the cysteine side
chain in a peptide or protein is generally around the mid 8s. So if you
keep your pH way down in the acid range, you'll greatly slow the reaction.
Similarly, whenever you want to react that cysteine thiol, jack the pH up to
at least 7. Note that at physiological pH (the mid 7s) thiols react pretty
quickly, indeed, so if you're looking at doing a biological sort of
experiment, you're going to end up with a lot of peptide disulfides (and
possibly mixed disulfides!) anyway.
Have fun!
Mark
-----Original Message-----
From: Thomas Weber [mailto:thomas.weber@mssm.edu]
Sent: Monday, May 14, 2001 4:20 PM
To: Recipients of ABRF List
Subject: PepSyn, Purification yields degradation products
Dear All,
I synthesized the following peptide with Fastmoc chemistry on rink amide
resin:
H W Y D S F V P W G H Q C
H, Q, C were protected as Trt
D,S,Y as tBu
After initial problems with deprotection (oxidation of the Trp
residue(s)) that I solved with your help and adding more and different
scavengers (thanks !) I went on to purify the peptide by RP-HPLC (C18,
0.1%TFA in water =A, 0.1% TFA in acetonitrile =B). According to the UV
trace this purification went very smoothly. However, when I rerun the
sample the sample seems to be actually less pure than the original crude
peptide (which was >90% pure). The additional peaks I see elute a little
later than the original peptide and look similar to the oxidation
products I observed earlier. Could it be that this is air oxidation ? I
kept the peptide as a lyophilized powder at -20°C over drierite. Any
suggestions what might be going on and how to prevent this degradation ?
Thanks
Thomas
-- Dr. Thomas Weber Institute for Gene Therapy and Molecular Medicine Box 1496 Mount Sinai School of Medicine 1425 Madison Avenue New York, NY 10029-6574 United States of AmericaPhone (office): (212) 659 8293 Phone (lab): (212) 659 8299 Fax: (212) 849 2437 e-mail: thomas.weber@mssm.edu
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