Examination of the protein binding behaviour of immobilised copper (II)-2,6-diaminomethylpyridine and its application in the immobilised metal ion affinity chromatographic separation of several human serum proteins.

A new metal ion chelator has been developed for use in the immobilised metal ion affinity chromatography (IMAC) of proteins. The aromatic tridentate ligand 2,6-diaminomethylpyridine (bisampyr), 1, was prepared as the dihydrochloride salt, via a two step synthesis from 2,6-pyridinedimethanol, 2, and immobilised onto Sepharose CL-4B through an epoxide coupling procedure. The resulting sorbent was chelated with Cu2+ ions to a density of 420 micromol Cu2+ ions per g gel and then characterised by frontal analysis using the protein, horse heart myoglobin (HMYO), at pH 7.0 and 9.0. From the resulting adsorption isotherms, the adsorption capacity, qm, for HMYO at pH 7.0 and pH 9.0 with the immobilised Cu2+-bisampyr Sepharose sorbent was found to be 1.27 micromol protein/g gel and 1.43 micromol protein/g gel, whilst the corresponding dissociation constants, K(D)s, were 18.0 x 10(-6) M and 16.0 x 10(-6) M respectively. The results confirm that the HMYO-Cu2+-bisampyr complex had similar stability at these pH values. This finding is in contrast with the situation observed with some other commonly used IMAC chelating ligates such as Cu2+-iminodiacetic acid (Cu2+-IDA) or Cu2+-nitrilotriacetic acid (Cu2+-NTA). Using human serum proteins, the interactive properties of the immobilised Cu2+-bisampyr Sepharose sorbent were further characterised at pH 5.0, 7.0 and 9.0 with specific reference to the binding behaviour of albumin, transferrin, and alpha2-macroglobulin.