Optimising the Purification of Terpyridine-Cytochrome c Bioconjugates

Joshua R. Peterson, and Pall Thordarson
* Author for corresponding; e-mail address: p.thordarson@unsw.edu.au
Volume: Vol.36 No.2 (MAY 2009)
Short Communication
DOI:
Received: -, Revised: -, Accepted: -, Published: -

Citation: Peterson J.R. and Thordarson P., Optimising the Purification of Terpyridine-Cytochrome c Bioconjugates, Chiang Mai Journal of Science, 2009; 36(2): 236-246.

Abstract

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.

Keywords: bioconjugate chemistry, cytochrome c, terpyridine, metalloterpyridine, protein purification, chromatography, FPLC, MALDI-TOF MS, Cation-exchange, IMAC, SEC, bioelectronics.

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