Importance of the α-amino group in the selective purification of synthetic histidine peptides by immobilised metal ion affinity chromatography

Abstract

The retention behaviour of some histidine containing peptides on Cu 2+- and Ni 2+-loaded immobilised metal ion affinity chromatography (IMAC) supports has been investigated and compared with that observed for the corresponding compounds lacking the free α-amino group and/or the imidazole function. On immobilised Cu 2+ all histidine-containing peptides, including those with a blocked α-amino function, were strongly retained above pH 5. The presence of a free α-amino group increased the retention marginally. On immobilised Ni 2+ histidine peptides with a free α-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 α-amino group. These observations indicate the involvement of two equipment attachment points in the binding. It seems that IMAC on a Ni 2+-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 α-amino group, which can be used as an affinity handle, will be present only on the target peptide.