Cysteine Blocking

Pranav Garg pgarg at tufts.edu
Thu Apr 15 16:27:10 EST 1999


I am working on a HPLC (Strong Cation Exchange) resolution of Lysozyme
variants (that vary by the oxidation state, with remaining cysteines
blocked; Lysozyme has 4 disulfide linkages in the Native form), or if
you will, quenched (= blocking of cysteines to prevent further
refolding) lysozyme intermediates that are obtained during the refolding

of a denatured and completely reduced lysozyme back into its native
structure.  I am trying to identify a reagent that will

1.  react "irreversibly", ie. not by a disulfide bond with the free
cysteines on the protein

2.  Impart a +ve charge on the cysteine site (thus increasing the
binding ability of the intermediate to
     the Cation Exchange Column, based on the number of cysteines
available for blocking)

3.  Have the highest possible mass that may help resolution using a
MALDI-TOF Mass Spec
     procedure.

So far, I have seen "ethylenimine" that satisfies criteria 1 and 2,
which is not bad, but ethylenimine seems to be out of commercial
availability due to its extreme carcinogenicity.

If anyone has come accross another good reagent for the purpose, that
would be helpful.

Thank You,

Pranav Garg
Biotechnology Center,
Department of Chem. Eng.
Tufts University
Medford, MA 02155

Phone 617 6273900
Fax     617 6273991
Email   pgarg at tufts.edu






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