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New α‐amino phenylalanine tetrazole ligand for immobilized metal affinity chromatography of proteins
Author(s) -
Lei Genhu,
Liu Liting,
Xiong Xiaohu,
Wei Yinmao,
Zheng Xiaohui
Publication year - 2008
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.200700520
Subject(s) - chemistry , tetrazole , sorbent , chromatography , phenylalanine , lysozyme , adsorption , dissociation constant , affinity chromatography , ligand (biochemistry) , chelation , dipeptide , metal ions in aqueous solution , metal , nuclear chemistry , organic chemistry , amino acid , enzyme , biochemistry , receptor
A new chelating compound has been developed for use in the immobilized metal affinity chromatographic (IMAC) separation of proteins. The bidentate ligand, α‐amino phenylalanine tetrazole, 4 , was synthesized via a five‐step synthesis from N ‐fluorenylmethoxycarbonyl phenylalanine and then immobilized onto silica through the epoxide coupling procedure. The binding behavior of the resulting IMAC sorbent, following chelation with Zn 2+ to a density of 183 μmol Zn 2+ ions/g silica, was characterized by the retention of proteins in the pH range of 5.0–8.0, and by the adsorption behavior of lysozyme with frontal chromatography at pH 6.0 and 8.0. The prepared column showed the separation ability to four test proteins and the retention time of these proteins increased with an increase in pH. From the derived isotherms, the adsorption capacity, q m , for the binding of lysozyme to immobilized Zn 2+ ‐α‐amino phenylalanine tetrazole–silica was found to be 1.21 μmol/g at pH 6.0 and 1.20 μmol/g sorbent at pH 8.0, respectively, whilst the dissociation constants K D at these pH values were 5.22×10 –6 and 3.49×10 –6 M, respectively, indicating that the lysozyme was retained more stable under alkaline conditions, although the binding capacity in terms of micromole protein per gram sorbent remained essentially unchanged.