Re: Non-specific cleavage for endoproteinase lys-c

Deb McMillen (mcmillen@morel.uoregon.edu)
Wed, 1 Apr 1998 17:25:47 -0800 (PST)

Joe, Hello,
I was wondering if you had any response to this email? We have a peptide
fragment that we would like to cleave between a C and an H--but we don't
have much of the peptide so only want to try it if it looks like it might
work. Thanks, Deb McMillen
Institute of Molecular Biology
University of Oregon

On Wed, 25 Feb 1998, Joseph Fernandez wrote:

> 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
>
>