Isolation, Purification, Some Properties And Modification Of Groups Of The Alcohol Dehydrogenase From Pig Liver

As one of the most important oxidordeuctase, Alcohol dehydrogenase(ADH,EC1.1.1.27) is widely existed in human and mammalian liver, plant tissues and microorganisms. It can be used to catalyze the oxidation of short-chain alcohols and aromatic alcohol to their corresponding carboxylic compounds along with the oxidized coenzyme I(NAD +) into reduced coenzyme I(NADH). ADH is widely used and has been applied in the many fields, such as clinical examination, chemistry and food industry. Currently, almost all of ADH products in the domestic market are derived from fermented yeast. It can not meet the needs of various fields because of its single source, and high-purity ADH depends heavily on import from abroad. Therefore,development of different sources of ADH with high purity and low cost has important significance in both theory and practice.Pig is one of the most commonly farming agriculture animal, and its genetic and physiological characteristics are highly similar to human beings. Hence, pig is recognized as the one of the most useful and versatile experimental animal model, and it is important for the research of agriculture and human disease. In this paper, we carried out a research on the isolation, purification of alcohol dehydrogenase from pig liver, as well as studied some properties and functional groups of alcohol dehydrogenase from pig liver. Our aim is to provide a new way of obtaining the ADH, and supply reference data for medical research and future in-depth study.1. Separation and Purification of ADH from Pig LiverElectrophoresis purity ADH from pig liver was obtained successively through homogenization, buffer extraction, ammonium sulfate fractionation precipitation,DEAE-Sepharose ion-exchange chromatography and Superdex-200 gel filtration chromatography. Results showed that the specific activity of ADH was 1 622.33 U/mg,recovery 29.05%, purification fold 34.58.2. Characteristics of ADH from Pig LiverThe molecular weight of ADH was approximately 171.79 kD, in which the subunit molecular mass was roughly 43.68 kD, According to the above results, we speculate that this enzyme is composed of four identical subunits. Besides, the study about enzymological properties of ADH showed that the optimum temperature and pH of this enzyme were 45 ℃ and 10.0 respectively. The enzyme was stable relatively in25~45 ℃ and pH 7.5~9.0, and in optimum conditions, its apparent Km towards ethanol was 19 mmol/L.The enzymatic activity of ADH could be strongly inhibited by chloroform, nbutanol, isopropanol, oxalic acid, sodium dodecyl sulfate(SDS), Cu2+, Zn2+, Ag+, while the enzymatic activity of ADH is activated by Mg2+. Furthermore, EDTA disodium has the dual role of this enzyme, when the concentration of EDTA disodium was less than 5mmol/L, the enzymatic activity was significantly increased, when the concentration was higher than 5 mmol/L, the enzyme activity had a certain downward trend.3. Chemical Modification of ADH from Pig LiverADH side chain groups were selectively modified by specific chemical modification agents. Firstly, serine, cysteine, tryptophan, methionine residues were essential function groups of ADH active center. Second, imidazole group of histidine may be the functional groups of the enzymatic active center. Thirdly, arginine guanidine,lysine and tyrosine residues have no direct relationship with ADH active center, and they are not the essential function groups of ADH.