Diamine Oxidase from Pig Kidney

Abstract

Abstract Diamine oxidase was prepared from pig kidney by a procedure involving heat denaturation at 60°, ammonium sulfate fractionation, pH precipitation, column electrophoresis, and chromatography on diethylaminoethyl cellulose. The procedure gave 3200-fold purification over the crude kidney cortex homogenate. The purified enzyme was homogeneous by the criteria of gel electrophoresis and ultracentrifugation. Some preparations showed a slower component of about 10%. The purified enzyme was pink-yellow and showed two absorption maxima, at 405 and 480 mµ. The latter absorption was diminished by addition of substrate under anaerobic conditions and restored by adding oxygen. No flavin was detected in the purified enzyme. The enzyme contained 11 to 12 mµmoles of copper per mg of protein. The diethyldithiocarbamate-treated enzyme was catalytically inactive. The activity was restored by adding suitable amounts of cupric copper; however, an excess of this metal inhibits the activity. The electron paramagnetic resonance spectrum is typical of Cu(II) in an environment of tetragonal symmetry. All copper atoms appear to be equivalent and in the cupric state. Addition of excess substrate did not lead to reduction of the copper, but changes in the electron paramagnetic resonance spectrum were observed which were specific for each individual substrate.