Purification and properties of coproporphyrinogenase

Abstract

Coproporphyrinogenase has been prepared from rat-liver mitochondria and its properties have been studied. The isoelectric point was around pH 5.0 and the molecular weight to be 80,000 [plus or minus] 8000. The pH optimum of the enzymic reaction was 7-4 and the apparent Km was of the order 0.03 m[image]. The enzyme was destroyed by boiling, and irreversible inactivation occurred below pH 3.5. It could be stored at[long dash]10[degree] without loss of activity. The enzyme acts specifically on coproporphyrinogen III and does not form pro-toporphyrinogen from trans-2,4-diacrylicdeuteroporphyrin or its porphyrinogen. It was unaffected by prolonged dialysis and no cofactor requirement could be demonstrated. Column chromatography of a partially purified enzyme preparation on Sephadex G-200 was found to be an improved method of purification, which gave a coproporphyrinogenase 58-fold purified. The purified enzyme was studied electro-phoretically but no evidence was obtained to suggest that more than one enzyme was involved in the reaction. The action was studied of various compounds added to the system. The presence of monothiol groups in the enzyme system was indicated, whereas vicinal dithiol groups were not involved in the reaction. Metalchelating agents did not inhibit the reaction, and no requirement for the presence of any essential metal has been found. All attempts to demonstrate the presence of a prosthetic group, in particular flavines, failed. Neither pyridoxal phosphate nor ATP was involved in the reaction, nor was a mitochondrial electron-transport chain required for the activity of the enzyme. Some circumstantial evidence was obtained to suggest that cis-2,4-diacrylicdeuteroporphyrin is an intermediate in the reaction.