New α-amino phenylalanine tetrazole ligand for immobilized metal affinity chromatography of proteins

✍ Scribed by Genhu Lei; Liting Liu; Xiaohu Xiong; Yinmao Wei; Xiaohui Zheng

• Publisher: John Wiley and Sons
• Year: 2008
• Tongue: English
• Weight: 698 KB
• Volume: 31
• Category: Article
• ISSN: 1615-9306
• DOI: 10.1002/jssc.200700520

✦ Synopsis

Abstract

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.