Thanks to all who responded to my question about how to get rid of H2O2 from protein solutions, in such a way that the protein would not be further modified. In summary, the suggestions were:
1. Gel filtration, either with prepoured columns or by FPLC.
2. Repeated microcentrifugal ultrafiltration (e.g. Nanosep or Microcon)
3. Addition of an excess of methionine.
4. Addition of catalase (References: N. Neumann, Methods in Enzymology, Volume XI, pp 485-487 (1967); FEBS Letters 455 247 (1999))
DTT would probably work, but there was some uncertainty about whether or not DTT would reduce methionine oxide in the protein. Any comments about this?
Now for my next question. After we isolate protein variants by rpHPLC (C4 or C18, Acetonitrile gradients, 0.1% TFA), we dry the solutions down by vacuum centrifugation, and then digest with trypsin for peptide maps. Sometimes we see almost complete oxidation of the methionine-containing peptides, but the extent of oxidition is erratic. How does one minimize this apparent air oxidation?
Vernon
Vernon A. Shoup
Regeneron Pharmaceuticals
Rensselaer, NY 12144
(518)488-6012
(518)488-6030 FAX
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