IMPORTANCE OF THE ALPHA-AMINO GROUP IN THE SELECTIVE PURIFICATION OF SYNTHETIC HISTIDINE PEPTIDES BY IMMOBILIZED 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 pept ides, including those with a blocked alpha-amino function, were strong ly retained above pH 5. The presence of a free alpha-amino group incre ased the retention marginally. On immobilised Ni2+ histidine peptides with a free alpha-amino group were strongly bound with a maximal reten tion at pH 8.5. Blocking of the amino group or removal of the imidazol e moiety reduced the maximal retention by a factor 5 to 10, with no re tention observed for peptides lacking both histidine and a free alpha- amino group. These observations indicate the involvement of two equipo tent attachment points in the binding. It seems that IMAC on a Ni2+-lo aded 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, whic h can be used as an affinity handle, will be present only on the targe t peptide.