Protocatechuate 3,4-Dioxygenase

A procedure is described for the purification and crystallization of protocatechuate 3,4-dioxygenase from p-hydroxybenzoate-induced cells of Pseudomonas aeruginosa. Overall purification was about 30-fold, with 23% yield. Crystallization of the enzyme was achieved when it was incubated with a sulfhydryl reagent such as mercaptoethanol for over 1 week prior to the addition of ammonium sulfate. The crystalline enzyme was homogeneous as judged by ultracentrifugation, starch gel electrophoresis, and immunoelectrophoresis. The molecular weight was estimated to be approximately 700,000 and the molecular activity was calculated to be 45,500 at 24°. The enzyme had a deep red color with a broad absorption between 400 and 650 mµ, and contained about 7 g atoms of nonheme iron per mole of enzyme. Crystallization was also achieved by treating the enzyme with iodoacetamide prior to the addition of ammonium sulfate, or by dialyzing it against distilled water. Crystalline enzyme obtained by these methods showed the same enzymic activity and physicochemical properties as described above. Polymerization of the enzyme appeared to occur under certain conditions and to prevent the formation of crystals. The mode of polymerization and certain other properties of the crystalline enzyme are also reported.