Non-specific cleavage for endoproteinase lys-c
Joseph Fernandez (fernaj@rockvax.rockefeller.edu)
Wed, 25 Feb 1998 09:23:52 -0500 (EST)
Over the past 3 years we have observed on two occasions possible
cleavage at carboxyamidomethyl cysteine (CAMC) using endoproteinase lys-c
from achromobacter. Last night we sequenced a peptide obtained after
digestion of a PVDF-bound protein (after reduction and
carboxyamidomethylation) that was found in the database using the BLASTp
search program. It ended up with a cysteine residue before the first
residue thus indicating a camc clip. Prior to these three years we did
not routinely reduce and alkylate cysteines and never observed a cys clip
before. The first two times we could never definitively prove the CAMC
clip, but this one has a lot of evidense. Here are my questions
1) Has anyone observed this non-specific cleavage before.
2) The R/A we perform is complete for most proteins (we have never observed
a peptide with a free cysteine since performing R/A) so most likely we have
alkylation. Another possibilty is the cys has been modified the acrylamide
cysteine and that is what is cleaving (although there were 2 CAMC's
observed in the peptide). What does anyone think of this?
3) The amino terminal residue in the sequenced peptide was HIS. Anyone
ever observe cleavage at the N-terminal side of HIS before? I do not
recall our earlier runs if there was a HIS or not.
4) Anyone know of any standard proteins low in lysines that could have a
CYS-HIS bond to check out the non-specificity?
5) One last thing is that WAKO's literature states that the enzyme cleaves
at S-aminoethylcysteine as well as lysine. Anyone know of a side reaction
of CAMC to form S-aminoethylcysteine or more likely acrylamide cysteine to
generate into S-aminoethylcysteine? Is there such a thing as ACRYLAMINE in
acrylamide?
Looking foreward to any answers, suggestions, opinions or useful or
humerous comments.
Joe
3)
Joseph Fernandez
Associate Director
The Rockefeller university
Protein/DNA Technology Center
1230 York Ave. New York, NY 10021
Phone: (212)-327-8869
FAX : (212)-327-8620
email: fernaj@rockvax.rockefeller.edu
Lab Web Page: http:\\pdtc.rockefeller.edu