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 meta l ion affinity chromatography (IMAC) of proteins. The aromatic tridentate l igand 2,6-diaminomethylpyridine (bisampyr), 1, was prepared as the dihydroc hloride 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 mu mol C u2+ ions per g gel and then characterised by frontal analysis using the pro tein, horse heart myoglobin (HMYO), at pH 7.0 and 9.0. From the resulting a dsorption isotherms, the adsorption capacity, q(m), for HMYO at pH 7.0 and pH 9.0 with the immobilised Cu2+-bisampyr Sepharose sorbent was found to be 1.27 mu mol protein/g gel and 1.43 mu mol protein/g gel, whilst the corres ponding 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 im mobilised Cu2+-bisampyr Sepharose sorbent were further characterised at pH 5.0, 7.0 and 9.0 with specific reference to the binding behaviour of albumi n, transferrin, and alpha(2)-macroglobulin. (C) 1999 Elsevier Science B.V. All rights, reserved.