Extensive investigation has been carried out to elucidate the mechanisms involved in pseudoligand affinity chromatography using textile dyes, and, empirically, it has been attributed to the chemical and steric structures of dye and protein. Possibly, a variety of interactions especially ionic and/or hydrophobic influence with a varying share in the binding and differ from protein to protein and from dye to dye. In this study, we have attempted to understand the effect of various biophysical parameters like the nature of the eluant, pH, and ionic strength on the binding of crude luteinizing hormone (LH) with various triazine-based dyes and thus predict their nature. Based on the elution patterns, cibacron and reactive brown suggested a dual electrostatic and hydrophobic nature. Reactive blue and reactive yellow reflected a major electrostatic/ionic nature with yellow offering 50-fold purification in a single step, while reactive red and reactive green had a predominant hydrophobic nature. Appreciably, reactive red was binding LH very tightly unlike other dyes, and addition of the arginine in the elution buffer substantially weakened the protein-dye interactions. pH was observed to be a principal factor assisting the protein-dye binding as well as hydrophobicity of the dye and the proteins. 1. Introduction Dye ligand chromatography is rapidly arising as a suitable substitute to specific affinity chromatographies owing to their easy accessibility and economical and effortless immobilization. Despite being synthetic in nature, the triazine-based dyes have a high affinity for many proteins and enzymes [1–7]. In the immobilized form, the dye has rapidly been identified and used as an affinity ligand in chromatographic isolation of a variety of proteins including dehydrogenases, kinases, serum albumin, interferon, several plasma proteins, and a host of other proteins owing to their ability to mimic the configuration of substrates, cofactors, or binding agents, thereby leading to high recovery and many a time purity in tandem, both achieved in a single chromatographic step [8–11]. Most of the dye and protein interaction studies have been performed on the blue sepharose (cibacron blue linked to beaded agarose) involving different dehydrogenase [12, 13]. The affinity ligands both synthetic as well as biological are being designed using a variety of procedures; however, an interesting alternative is presently being offered in the form of biomimetic dyes or chimaeric dye-ligands involving a chloro-triazinlyl scaffold which is often replaced with various amino
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