Re: cysteines

Dan Crimmins (crimmins@pathbox.wustl.edu)
Fri, 27 Mar 1998 08:25:10 -0600 (CST)

At 07:22 PM 3/26/98 -0500, john holt wrote:
>Does anyone have an idea how I might keep cysteines reversibly reduced
>during a purification using reverse phase HPLC under the usual 0.1% TFA
>conditions?? I know traditional alkylation works, and allows me to achieve
>the purification, and I know reduction and hoping
>-that-reoxidation-won't-occur-upon-lowering-the-pH and proceeding, doesn't.
>
>John Holt
>
>Pasteur Merieux Connaught
>
>
>
>

John,
Try using Ellman"s reagent (DTNB) at a high molar ratio of rgt to
thiols to avoid intra- /inter- disulfide crosslinking of peptide thiols.
The mixed disulfide adduct is stable to typical RP-HPLC conditions and as a
bonus absorbs at 325 nm so you can quantitate the number of adducts if
desired. To remove the TBN moiety simply reduce with DTT at neutral pH.
Both reactions, the addition step and removal, are virtually quantitative.
You can check for removal by monitoring the TBN anion at 410 nm.

Regards,

Dan L. Crimmins
Washington University School of Medicine
Dept. Pathology/Division of Laboratory Medicine
660 S. Euclid Ave., Box 8118
St. Louis, MO 63110
Phone: 314-454-8514; Fax: 314-454-5208
e-mail: crimmins@labmed.wustl.edu