Protein Protocols Protein Protocols

Cleavage at Tryptophanyl-x Bonds 493
72
Chemical Cleavage of Proteins at Tryptophanyl-X
Peptide Bonds
Bryan John Smith
1. Introduction
Tryptophan is represented in the genetic code by a single codon and has proven
useful in cloning exercises in providing an unambiguous oligonucleotide sequence as
part of a probe or primer. It is also one of the less abundant amino acids found in
polypeptides, and cleavage of bonds involving tryptophan generates large peptides.
This may be convenient for generation of internal sequence information (the usual
purpose to which this technique is put), but is less useful for identification of proteins
by mass mapping, where a larger number of smaller peptides makes for more successful
database searching.
While there is no protease that shows specificity for tryptophanyl residues, various
chemical methods have been devised for cleavage of the bond to the carboxy-(C)terminal
side of tryptophan. These are summarized in Table 1. Some show relatively
poor yields of cleavage, and/or result in modification of other residues (such as irreversible
oxidation of methionine to its sulfone), or cleavage of other bonds (such as
those to the C-terminal side of tyrosine or histidine). The method described in this
chapter is one of the better ones, involving the use of cyanogen bromide (CNBr).
Cleavage of bonds to the C-terminal side of methionyl residues (see Chapter 71) is
prevented by prior reversible oxidation to methionine sulphoxide. Cleavage by use
of N-bromosuccinimide or N-chlorosuccinimide remains a popular method, however,
despite some chance of alternative reactions (see Table 1).
Apart from its use in peptide mapping and generation of peptides for peptide
sequencing, cleavage at tryptophan residues has also been used to generate peptides
used to map the binding site of an antibody (1), or the sites of phosphorylation (2).
Another application has been generation of a recombinant protein from a fusion protein
(3): tryptophan was engineered at the end of a β-galactosidase leader peptide, adjacent
to the N-terminus of phospholipase A2. Reaction with N-chlorosuccinimide allowed
subsequent purification of the enzyme without leader peptide.
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
493