Purification of cysteine-containing synthetic peptides via selective binding of the alpha-amino group to immobilised Cu2+ and Ni2+ ions.
Peptides containing a cysteine residue but lacking histidine and tryptophan were synthesised by the solid-phase method. Their retention behaviour on Cu2+ - and Ni2+ -loaded immobilised metal ion affinity chromatography (IMAC) supports at pH 5-11 was studied and compared with that observed for the corresponding compounds without the free alpha-amino group and/or the thiol function. Unexpectedly, it was found that neither a cysteine side-chain nor a cysteine disulphide affects the retention of the peptides. A free alpha-amino group is required for binding; no retention is observed in its absence. At pH 9 substantial amounts of metal ions were transferred from the chromatographic support to an alpha-amino-protected cysteine-containing peptide. However, at pH 7 no such transfer occurred. Therefore, the lack of retention observed for peptides with a blocked alpha-amino function over the entire pH range is not solely caused by metal ion scavenging by the thiol group. Partial dimerisation may occur upon chromatography; the dimers formed are retained strongly due to the presence of two free alpha-amino groups. It seems that IMAC on a Cu2+ - or Ni2+ -loaded support can be used for the purification of cysteine-containing peptides synthesised by the solid-phase method. Inclusion of a capping protocol in the synthesis ensures that a free alpha-amino group, which can be used as an affinity handle, will be present only on the target peptide.
Hansen
P
P
School of Biochemistry, La Trobe University, Bundoora, Australia.
Andersson
L
L
Lindeberg
G
G
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods; D003300:Copper / Q000378:metabolism*; D003545:Cysteine / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009532:Nickel / Q000378:metabolism*; D010455:Peptides / Q000302:isolation & purification* / Q000378:metabolism
D010455:Peptides; D003300:Copper; D009532:Nickel; D003545:Cysteine