METHOD OF LACTATE DEHYDROGENASE PREPARING Russian patent published in 2002 - IPC

FIELD: biochemistry, biotechnology, enzymes. SUBSTANCE: porcine muscles are used as the parent raw. Muscles are homogenized, suspended in 1 volume of water and centrifuged. Enzyme is precipitated with ammonium sulfate at 54-56% of saturation, centrifuged and enzyme precipitate is dissolved in buffer, pH 6.0-7.0, followed by centrifugation, dialysis against the same buffer and final centrifugation. Enzyme is subjected for purification by chromatography on sulfopropyl-Sephadex as cation- exchanger in 0.03-0.05 M potassium-phosphate buffer, pH 6.0-7.0, at concentration gradient of potassium chloride from 0.056 M to 0.13 M. Lactate dehydrogenase is precipitated with ammonium sulfate at 52-55% of saturation, centrifuged, enzyme precipitate is dissolved in buffer and stored as suspension in 52-55% ammonium sulfate solution. Method provides preparing highly purified lactate dehydrogenase with specific activity up to 330 U/mg of protein and yield up to 70%. Impurity of pyruvate kinase in the final preparation is 0.04%, not above, and impurity of asparatate- and alanine aminotransferase is 0.007%, not above. Invention can be used for enzyme activity assay in biological fluids. EFFECT: simplified process of enzyme preparing, increased specific activity and yield, enhanced purity. 4 cl