Simple isolation method of glutamate dehydrogenase from pig liver using dye ligand chromatography and dye ligand membranes

Poster presented on the 9^th European Congress on Biotechnology, Brussels, 12–15 July 1999, Abs 1526.

Simple isolation method of glutamate dehydrogenase from pig liver using dye ligand chromatography and dye ligand membranes.

Miklós PÉCS, Tamás DÉVÉNYI. Technical University Budapest, Dept. of Agricultural Chemical Technology, 1521 Budapest, POB 91, Gellért tér 4., Hungary

Affinity chromatography is a widely used isolation method of different molecules with different biochemical activities. Dye ligand chromatography is a special way of affinity chromatography utilising the structural analogy between the nucleotide cosubstrates (NAD, NADP, ATP) and the triazine dyes (Cibacron serial, Procion serial) used earlier as reactive dyes in the textile industry. The selectivity of these ligands is not so good as e.g. the antigen-antibody interaction because a large number of enzymes have a nucleotide binding site. About hundred different enzymes – mainly oxidoreductases - were investigated in dye chromatography experiments. Glutamate dehydrogenase is an enzyme of commercial importance (diagnostics, etc.) was hardly mentioned in dye ligand chromatography publications. The standard isolation procedure has twelve steps (salt and heat precipitation, centrifugation, etc.) and our goal was to simplify the technology and to reduce the number of steps with dye ligand chromatography. At first the equilibrium of the GlDH – Cibacrone Blue-agarose system was investigated. Adsorption isotherm and constants were determined in batch experiments. Than the parameters of adsorption and elution (pH, ionic strength, elution profile) were determined and optimised with pure GlDH enzyme preparate. At the end the enzyme was isolated from pig kidney, more than 90% of the total activity was recovered, the purification factor was above 80. Best purification results were achieved with dye ligand activated adsorption membranes instead of column but the capacity of this system was lower. The optimised isolation procedure had only five steps and the purity (specific activity) of the preparate was higher than of the standard product.