Multivalent binding interaction of alcohol dehydrogenase on dye-metal affinity matrix

The optimization of a chromatographic process using immobilized metal affinity chromatography requires an understanding of the factors that govern the interaction between proteins and immobilized metal ions. Factors, such as concentrations of protein, NaCl and imidazole were investigated to elucidate kinetics of adsorption of alcohol dehydrogenase (ADH) onto a dye-iminodiacetic acid matrix (dye-IDA matrix). The results indicate that the adsorption of ADH onto a dye-IDA matrix occurs in the mode of multiple-site binding interactions between ADH and zinc ions immobilized on the dye-IDA matrix. The estimated average number of interaction sites was 4.5 and the association constant was 6 x 10(-9) mM(-n). The isotherm of ADH adsorption was well represented by a multivalent model of protein-zinc ion interactions. For the adsorption of ADH from clarified yeast homogenate, addition of imidazole as a protein competitor to adsorption buffer increased the adsorption specificity of ADH, thereby suppressing contaminant protein adsorption. It was also observed that the adsorption of ADH was better performed at high initial protein concentrations in the yeast homogenate. Consequently, these results may have important implications on the optimization of the strategy for immobilized metal affinity adsorption in packed and expanded bed systems.