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

doi:10.1016/0021-9673(94)01100-S

Journal of Chromatography A

Volume 690, Issue 2, 27 January 1995, Pages 155-159

https://doi.org/10.1016/0021-9673(94)01100-S Get rights and content

Abstract

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 α-amino group and/or the imidazole function.

On immobilised Cu²⁺ 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 Ni²⁺ 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 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 α-amino group, which can be used as an affinity handle, will be present only on the target peptide.

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¹: Present address: School of Biochemistry, La Trobe University, Bundoora 3083, Victoria, Australia.

^∗: Present address: Department of Medical and Physiological Chemistry, BMC, Box 575, S-751 23 Uppsala, Sweden.

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

Abstract

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