We have had great luck with peracetic acid at micromolar levels (see Warne et al. 1993 Pharm. Res. 10:5) 0.25 x to 2 x Met conc. gives only the sulfoxide at varying % modification. We have run this reaction for 1-2 hrs in various formulation buffers with 3 proteins. You must take care to use fresh peracetic acid or at least test it first on a less valuble peptide. One bottle we had was about 10 fold less reactive after sitting in the hood for 4 months.
Tom
***********************************************
Thomas J. Porter, Ph.D.
Principal Scientist
Biopharmaceutical Characterization
and Analysis
Genetics Institute
1 Burtt Road
Andover MA 01810
978-247-2166 (voice)
978-247-2837 (fax)
tporter@genetics.com
>>> Rod Levine <rlevine@nih.gov> 05/30 6:15 PM >>>
>At 04:19 PM 5/30/2000 +0200, Hanno Steen wrote:
> >Hi there, is there any protocol available to oxidise the methionine residue
> >in peptides in a controlled way so that only the sulfoxide is obtained. I
> >tried hydrogenperoxide (3%) and performic acid but in both cases I ended up
> >with the sulfonyl compound (the dioxide). Any suggestions are appreciated.
> >Thanks in advance, Hanno
Hanno,
I believe that performic will usually give you the sulfone, as well as
oxidizing cysteine and cystine to cysteic acid.
However, our experience with a number of proteins with hydrogen peroxide
has been rather good -- generating only the sulfoxide. We have
demonstrated that to be the case by amino acid analysis and, for some of
the proteins, by HPLC/mass spectroscopy. The various methionines
definitely differ in susceptibility to oxidation, so that you may wish to
examine both the concentration and time dependence of oxidation.
But, for a "single shot" experiment, I recommend trying 50-100 mM hydrogen
peroxide (somewhat less than you were using) for 1-2 hours at 37 C. We
tend to work in phosphate buffer for simplicity, but most other buffers are
fine as well. If your protein will tolerate it, a lower pH (say 5.0) is
preferable to neutral pH in order to avoid oxidation of other residues. It
is usually a good idea to include a chelator such as DETAPAC to avoid other
reactions, although if you do work at the lower pH you are not likely to
have much in the way of side reactions.
Rod Levine
NIH
Bldg 3, Room 106 MSC 0320
Bethesda, MD 20892-0320
email: rlevine@nih.gov
voice: 1 (301) 496-2310
fax: 1 (301) 496-0599
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