All of the cysteine proteinases (papain, actinidin, bromelain and dozens of
others) have single cysteines in their active sites that are
super-reactive, at least to smaller thiol reagents (such as iodoacetate,
dipyridyl disulfide, etc). The problem is that these enzymes are proteases
and might chew up your protein/peptide. Here's an idea, albeit untested,
to get around this problem. Treat papain (or bromelain; both a lot cheaper
than carbonic anhydrase) with ethylene sulfide (Aldrich) to generate
mercaptoethyl-papain (then dialyze or gel filter):
S
/ \
Papain-SH + H2C-CH2 ------> Papain-S-CH2-CH2-SH.
Alkylation of the active site thiol group will kill the protease activity
and generate a new thiol group which should be even more accessible for
reaction. One possible disadvantage of this approach is that it might be
smelly, since ethylene sulfide is stabilized with butyl mercaptan.
Richard Laursen
------------------------------------
>Dear friends:
>We are looking for carrier proteins having a single (and accessible)
>Cys residue. A database search seems to suggest human carbonic
>anhydrase 1 (HCA1) may be such a protein. The Sigma catalog (C4396,
>purified , lyophilized; and C5290, partially synthesized from C4395,
>about 2.5 times more expensive) does not specify this information,
>which we haven't succeeded in obtaining from Sigma either. Can any of
>the protein experts out there tell me whether the protein a) is
>monomeric, and ii) the thiol group is available? As far as I've been
>able to ascertain, the zinc atom in HCA1 is histidine, not
>cysteine-bound. Thanks a lot for any help.
>
>David
>
>--
>David Andreu
>Scientific Director; Peptide Facility
>University of Barcelona, Spain
>Phone/Fax: 3493-402 1260
>andreu@admin.qo.ub.es
Richard A. Laursen
Department of Chemistry
Boston University
590 Commonwealth Ave.
Boston, MA 02215
Tel (617) 353-2491; FAX (617) 353-6466
email: <laursen@bu.edu>