New ligand, N-(2-pyridylmethyl)aminoacetate, for use in the immobilised metal ion affinity chromatographic separation of proteins

A new chelating compound has been developed for use in the immobilised meta l ion affinity chromatographic separation of proteins. The tridentate ligan d, sodium N-(2-pyridylmethyl)aminoacetate (carbpyr), I, was prepared via a one-step synthesis from 2-picolylamine, 3 and then immobilised onto Sepharo se CL-4B through the epoxide coupling procedure. The binding behaviour of t he resulting IMAC sorbent, following chelation with Cu2+ ions to a density of 152 mu mol Cu2+ ions/g gel was characterised by frontal analysis experim ents using horse heart myoglobin (HMYO) at pH 7.0 and pH 9.0. From the deri ved isotherms, the adsorption capacity, q(in), for the binding of HMYO to i mmobilised Cu2+-N-(2-pyridylmethyl)aminoacetate (im-Cu2+-carbpyr)-Sepharose CL-4B at these pH values was found to be 1.92 and 1.91 mu mol/g sorbent, r espectively, whilst the dissociation constants K-D were 0.0092.10(-6) M and 0.0062.10(-6) M at pH 7.0 and pK 9.0, respectively, indicating that the HM YO-im-Cu2+-N-(2-pyridylmethyl)aminoacetate complex was more stable under al kaline conditions, although the binding capacity in terms of mu mol protein /g gel remained essentially unchanged. The selectivity features of the im-C u2+-carbpyr-Sepharose CL-4B sorbent were further characterised in terms of the binding properties with several human serum proteins at pH 5.0, pH 7.0 and pH 9.0. (C) 1999 Elsevier Science B.V. All rights reserved.