Rapid screening of textile dyes employed as affinity ligands to purify enzymes from yeast

RAYA-TONETTI, G; PEROTTI, N

BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY

PORTLAND PRESS LTD

Lugar: London; Año: 1999 vol. 29 p. 151 - 156

0885-4513

ABSTRACT: A rapid method for screening potential dye ligands for use in affinity chromatography is described. Textile dyes were non-covalently coupled to a cross-linked polysaccharide Sepharose(R) matrix. Yeast alcohol dehydrogenase (ADH) was used as the model protein for evaluating the screening system. A homogenate from baker´s yeast was used as the crude source of enzyme. Batchwise adsorption and elution were used to evaluate the individual dyes. The influence of pH and ionic strength in the binding and elution steps was evaluated. Batch isotherms were used to evaluate parameter characteristics. Experimental data obtained were fitted to Langmuir isotherms to determine the maximum binding capacity and the dissociation constant for each dye evaluated in this study. A dynamic binding capacity of 107.6 units of ADH/g of resin was determined for Procion Turquoise MXG dye by frontal analysis. Specific elution with NAD+ and non-specific elution with 50 mM Tris/HCl buffer, pH 8.5, were tested when Procion Turquoise MXG was used, giving purification factors of 53.5 and 4.4 respectively. This screening technique is inexpensive and can be performed in a few hours. It was possible to predict the performance of different reactive dyes in this way, and the influence of pH and salt on the binding behaviour was demonstrated.