Purification andProperties ofCoproporphyrinogenase

hasbeenprepared fromrat-liver mitochondria and itsproperties havebeenstudied. Theisoelectric point was foundtobearound pH15.0 andthemolecular weight tobe80000 + 8000.ThepH optimumofthe enzymicreaction was 7-4andtheapparentKm was oftheorder003mM. The enzyme was destroyed byboiling andirreversible inactivation occurred below pH3.5.Itcouldbestored at - 10°without lossofactivity. Theenzyme acts specifically on coproporphyrinogen IIIanddoesnotformprotoporphyrinogen fromtrans-2,4-diacrylicdeuteroporphyrin oritsporphyrinogen. Itwas unaffected byprolonged dialysis andno cofactor requirement couldbedemonstrated. 2. Columnchromatography ofa partially purified enzyme preparation on Sephadex G-200was foundtobean improved methodofpurification, whichgave a coproporphyrinogenase 58-fold purified. Thepurified enzyme was studied electrophoretically butno evidence was obtained tosuggest thatmore thanone enzyme was involved inthereaction. 3.Theaction was studied ofvarious compounds addedtothesystem.Thepresenceofmonothiol groupsintheenzyme system was indicated, whereas vicinal dithiol groups were notinvolved inthereaction. Metalchelating agentsdidnotinhibit thereaction andno requirement forthepresence ofany essential metalhasbeenfound.Allattempts todemonstrate thepresence ofa prosthetic group,inparticular flavines, failed. Neither pyridoxal phosphate nor ATP was involved inthereaction, nor was a mitochondrial electron-transport chainrequired fortheactivity oftheenzyme. Somecircumstantial evidence was obtained tosuggest thatci8-2,4-diacryli edeuteroporphyrin isan intermediate in thereaction. The penultimate stepofhaem biosynthesis, namelytheconversion ofcoproporphyrinogen III