Importance of the alpha-amino group in the selective purification of synthetic histidine peptides by immobilised metal ion affinity chromatography.
The retention behaviour of some histidine containing peptides on Cu2(+)- and Ni2(+)-loaded immobilised metal ion affinity chromatography (IMAC) supports has been investigated and compared with that observed for the corresponding compounds lacking the free alpha-amino group and/or the imidazole function. On immobilised Cu2+ all histidine-containing peptides, including those with a blocked alpha-amino function, were strongly retained above pH 5. The presence of a free alpha-amino group increased the retention marginally. On immobilised Ni2+ histidine peptides with a free alpha-amino group were strongly bound with a maximal retention at pH 8.5. Blocking of the amino group or removal of the imidazole moiety reduced the maximal retention by a factor 5 to 10, with no retention observed for peptides lacking both histidine and a free alpha-amino group. These observations indicate the involvement of two equipotent attachment points in the binding. It seems that IMAC on a Ni2(+)-loaded support can be used for the purification of histidine 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
Department of Immunology, University of Uppsala, Sweden.
Lindeberg
G
G
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D003300:Copper; D006639:Histidine / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009532:Nickel; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*
D010455:Peptides; D006639:Histidine; D003300:Copper; D009532:Nickel