Isolation, Purification, Some Properties And Modification Of Groups Of The Lactate Dehydrogenase From Duck Liver

Lactate dehydrogenase (LDH) is widely distributed in plants, animals and microorganisms. It is one of the important enzymes for the glycolysis metabolism of organisms, and mainly catalyze the reversible transforation between the Pyruvic acid and lactic acid. LDH in animals mainly exists three subunits H, M and C, H and M subunits composite five kinds of isozymes:LDH1(H4), LDH2(H3M), LDH3(H2M2), LDH4(HM3) and LDH5 (M4), LDH-C4 is the four polymers composed C subunit.LDH is widely used in the field medical diagnostic, it is the main production raw material of anine transaminase measurement kit, the verification of pyruvic acid salt, diagnostic of leukemia, liver disease, myocardial infarction pulmonary embolism, it is also used food additives of fermented dairy products, pickled products. So far, there are a lot of researches about LDH isozymes from different organisms except duck liver. So we determined to separate and purify the LDH from duck liver which is easy and cheap to obtain. We researched on the partial enzymatic characteristics of the enzyme. Our works aimed at providing reference for the deeply investigation and Industrial production of LDH. The results of study were follows:1. Separation and Purification of LDH from Duck LiverElectrophoresis-purity lactate dehydrogenase (LDH) from duck liver was obtained through the procedures of homogenization, buffer solution extraction, ethanol deposit, ammonium sulfate precipitation, DEAE-Sepharose ion exchange chromatography and Superdex-200 gel filtration chromatography.25.79% of the LDH activity was obtained. The specific activity was 668.82 U/mg. The purification fold of the LDH was 185.78.2. Characteristics of LDH from Duck LiverThe results of the study show:all molecular mass of the LDH was 170.93 kD. The subunite relative molecular mass of the LDH was 44.72 kD. The optimum temperature and pH of the LDH was 45 ℃ and 7.4, respectively. It was respectively stable in the range of pH 5～10 and 25～45 ℃. Its Km was 3.407 μmol/L towards NADH,8.431 μmol/L towards sodium pyruyate.Activity could be obviously inhibited when interacting with Cu2+, Ag+, chloroform, methanol, isopropanol, ethanol, oxalic acid, SDS, EDTA. Enzyme activity could be enhanced by low concentration of Co2+, K+.3. Chemical Modification of LDH from Duck LiverThe results of the study showed that:Serine, sulfydryl, tryptophan and methionine residues was essential function group of the enzyme activity center of LDH from duck liver. His, Lys, disulfide bonds, Arg, Tyr residues should be not essential function group of activity center of duck liver LDH.