Partial purification, characterization and mechanism study of sulfhydryl oxidase from bovine skim milk = Bovine skim milk sulfhydryl oxidase 의 부분정제, 특성 및 효소 반응 메카니즘에 관한 연구

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Sulfhydryl oxidase (EC was purified partially by Sephadex G-100 and DEAE -cellulose column chromatography was purified about 1800 fold over the skim milk and showed optimum pH at 7.2 and optimum temperature at 37$^\circ$C. The activation energy of the enzyme was calculated to be 13.8 Kcal/mole by the Arrhenius equation. The molecular weight of the enzyme was verified to be 87,000 using Sephadex G-200 column chromatography. And it was confirmed that the enzyme acted on the conversion of sulfhydryl groups to their corresponding disulfides. This enzyme was inhibited by the addition of metal chelator, EDTA, but not by o-Phenanthroline,$\alpha,\alpha``$ -Dipyridyl which are iron specific chelators. And this enzyme requires $Mn^{++}$ for its activity. The application of functional group blocking agents and the photooxidation didn``t show the involvement of imidazole or hydroxyl groups in the active site of the enzyme. The enzyme obeyed Michaelis-Menten type kinetics at the low substrate concentrations but at the high substrate concentration above 0.83mM of GSH, substrate inhibition was observed. The Km and Vmax values were calculated to be $1.0\times10^{-4}$ M and 4.0 units per milligram protein, respectively. And this enzyme showed broad substrate specificity for thiol compounds bearing over two functional groups. Plots of 1/vo vs. 1/(cyst.) at various concentrations of fixed substrate $O_2$ showed uncompetitive type. And this enzyme was not inhibited by a product, disulfides, under saturated $O_2$ concentration. These results represent that the complex of enzyme-substrate, from which disulfides depart, leaving hydrogenated enzyme. Oxygen then combines with the hydrogenated enzyme to give the complex of two, which decomposes to yield the product $H_2O_2$ and free enzyme.
Lee, Hyun-Jae이현재
한국과학기술원 : 생물공학과,
Issue Date
62414/325007 / 000771138

학위논문 (석사) - 한국과학기술원 : 생물공학과, 1979.2, [ vii, 62 p. ]


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