Benzothiazolium Salt as Chromatography Ligand for Protein Purification

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.