Protein Protocols Protein Protocols

Cleavage at Asparaginyl-Glycyl Bonds 507
75
Chemical Cleavage of Proteins at Asparaginyl-Glycyl
Peptide Bonds
Bryan John Smith
1. Introduction
Reaction with hydroxylamine has been used to cleave DNA and to deacylate proteins
(at neutral pH; see Note 6). At alkaline pH, however, hydroxylamine may be used
to cleave the asparaginyl-glycyl bond (see Note 1). This cleavage tends to generate
large peptides, as this pairing of relatively common residues is relatively uncommon,
representing about 0.25% of amino acid pairs, according to Bornstein and Balian (1). It
is therefore generally not useful for identification of proteins by mass mapping, which
is better served by larger numbers of peptides. It may be a useful method for further
cleavage of large peptides.
Cleavage at Asn-Gly bonds has been used for:
1. Generation of peptides for sequencing purposes (e.g., ref. 2).
2. Generation of peptides for use in mapping ligand binding sites (e.g. ref. 3) and phosphorylation
sites (e.g. ref. 4).
3. Identification of sites of succinimide (cyclic amide) sites in proteins (5).
4. Cleavage of a fusion protein at the point of fusion of the constituent polypeptides (although
it was noted that formation of hydroxamates may occur [6], as may minor cleavage
reactions [3]).
2. Materials
1. Cleavage buffer: 2 M hydroxylamine·HCl,
2 M guanidine·HCl,
0.2 M KCO3, pH 9.0
Use Analar grade reagents and HPLC grade water (see Note 2).
Beware the mutagenic, toxic and irritant properties of hydroxylamine. Wear protective
clothing. Clear wet spillages with absorbent material or clear dry spillages with a
shovel, and store material in containers prior to disposal.
2. Stopping solution: Trifluoroacetic acid, 2% (v/v) in water (high-performance liquid chromatography
[HPLC] grade).
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
507