Abstract
A new chromatography support was synthesized from Sepharose CL-6B containing a benzothiazolium bromide salt as a ligand. The original goal of this study was the evaluation of the azolium salt individual contribution as a constituent part of thiacarbocyanine dyes in an affinity or multi-modal relationship with standard proteins. This dye was previously post-grafted onto beaded cellulose by a curing method and successfully used as an affinity ligand for the interaction and isolation of different proteins. Following this idea, the immobilization of an azolium bromide salt onto Sepharose CL-6B in different concentrations was performed by Steglich esterification, using dicyclohexylcarbodiimide as a coupling reagent and 4-dimethylaminopyridine as basic catalyzer. The obtained crafted beaded Sepharose supports were qualitatively and quantitatively characterized by scanning electron microscopy, energy dispersive X-ray spectroscopy and elemental analysis. The interactions between the chromatographic supports obtained and several standard proteins were finally evaluated. The best results were achieved using a chromatographic support with a ligand density of 0.18 mmol of benzothiazolium salt/g of chromatography support, enabling the separation of ribonuclease, α-chymotrypsin and bovine serum albumin from an artificial mixture using a decreasing ammonium sulfate stepwise gradient. In conclusion, this study demonstrates the potential applicability of small multi-modal cationic ligands such as benzothiazolium salts, compared to these congener cyanine dyes previously studied, for the separation and purification of proteins by chromatography, revealing a distinct and useful chromatographic behavior by itself.
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Acknowledgments
The authors would like to thank the Portuguese Foundation for Science and Technology, Programa Operacional “Ciência, Tecnologia, Inovação”—POCTI and Fundo Europeu de Desenvolvimento Regional—FEDER, for the funding of the Project “PTDC/QUI–QUI/100896/2008” and PEst-C/SAU/UI0709/2011 COMPETE (Programa Operacional Factores de Competitividade). L. P. Alves acknowledges a fellowship (BI-1-PTDC/QUI–QUI/100896/2008) in the ambit of this project. We are also grateful to the Optical Centre of University of Beira Interior for the acquisition of the SEM and SEM–EDS images.
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Alves, L.P., Ramos, S.S., Sousa, F. et al. A Benzothiazolium Salt as Chromatography Ligand for Protein Purification. Chromatographia 77, 1597–1605 (2014). https://doi.org/10.1007/s10337-014-2774-x
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DOI: https://doi.org/10.1007/s10337-014-2774-x