On the nature of immobilized tris(carboxymethyl)ethylenediamine

Immobilized tris(carboxymethyl)ethylenediamine (TED) (also known as ethylenediamine-N,N,N'-triacetic acid) serves as a stationary phase for fractionation of proteins and the preparation of metal-free proteins by immobilized metal-ion affinity chromatography. The Cu(II) complex of commercially available immobilized TED has been characterized by elemental, electrochemical and spectroscopic techniques. There is a large discrepancy between the theoretical capacity determined from the nitrogen content and the experimental capacities determined by atomic-absorption spectroscopy and anodic stripping voltammetry (ASV), indicating that a substantial portion of the immobilized ligand is not binding Cu(II). In addition, the titration of immobilized TED with Cu(II), monitored by ASV, suggests that more than one ligand is involved in binding Cu(II). A comparison of the EPR spectrum for immobilized Cu(II)-TED with spectra for various model complexes shows that the major immobilized ligand is ethylenediamine-N,N'-diacetic acid (EDDA) with immobilized TED present only as a minor component. The complexation constant for Cu(II) is close to the value for Cu(II)-EDDA in solution. The formation of EDDA is consistent with the method for synthesizing immobilized TED.