MURINE LIVER HOMOGENTISATE 1,2-DIOXYGENASE - PURIFICATION TO HOMOGENEITY AND NOVEL BIOCHEMICAL-PROPERTIES

1. The murine liver enzyme homogenisate 1,2-dioxygenase (HGO) is purif ied 330-fold. The molecular mass, as determined by gel filtration, is approximately 149 kDa. SDS/PAGE under strongly reducing conditions rev eals a single band at 49 kDa, whose concentration increases during the purification. 2. KGO has a pI = 8. The pH optimum for activity in vit ro is 6.1. 3. The K-m for its natural substrate HGA is 188 mu M, where as the K-d for the ferrous ion is determined at 19 mu M. Fe2+ is an ab solutely obligate cofactor and cannot be replaced by other divalent ca tions. Incubating the enzyme with Fe2+ plus one of other divalent cati ons Zn2+, Cu2+, Co2+ or Ni2+ in equimolar concentrations inhibits HGO, whereas addition of Ca2+ and Mn2+ has no effect. Ascorbate probably a cts as a reducing agent, protecting Fe2+ from spontaneous oxidation or by reversing its oxidation. Ascorbate has a great potential to stabil ize the assay system. 4. The activation energy determined by an Arrhen ius plot is 24 kJ/mol. 5. HGO consists of a single type of subunit wit h no intermolecular disulfide bridges and requires Fe2+ as a cofactor. This emphazises that the enzyme is familiar with the class of extradi ol dioxygenases.