Chiang Mai Journal of Science

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The synthesis and purification of a terpyridine cytochrome c (tpy-cyt) bioconjugate is reported. Following previously reported procedures, crude cytochrome c was successfully purified by strong-cation exchange chromatography, however, this method was found to be unsuccessful for the purification of  tpy-cyt bioconjugates. Similar results were obtained with weak-cation exchange chromatography. The results obtained also suggested that metalcoordination to the terpyridine ligand in  tpy-cyt bioconjugates adversely affects their interaction with cation-exchange column media. Attempts to utilize the affinity of terpyridines for zinc(II) ions to form tpy-cyt dimer that could subsequently be purified by size-exclusion chromatography were unsuccessful, however, added further evidence to the role of metal-ion coordination to the chromatographic properties of  these bioconjugates. Based on these results, a method using nickel(II) loaded immobilized metal-ion affinity chromatography (IMAC) was then successfully developed and employed to obtain the tpy-cyt bioconjugate in good purity. These results will be of  significant importance to us and others that are developing redox-protein based bioconjugates that incorporate metal-chelating ligands, such as terpyridine, for application in bioelectronic devices.

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