THE ENZYMIC INCORPORATION OF IRON INTO 

 PROTOPORPHYRIN 



By R. A. Neve* 



Department of Biochemistry, University of California, 

 Berkeley, California 



Following the elegant studies on porphyrin biosynthesis, interest has 

 centred on the incorporation of iron into protoporphyrin to form haem. 

 There is evidence that iron may be complexed non-enzymically by copro- 

 porphyrinogen and protoporphyrinogen in aqueous solutions, and at 

 physiological pH and temperature (Neilands and Orlando, 1957; Heikel, 

 Lockwood and Rimington, 1958). However there is now a considerable 

 amount of data demonstrating the enzymic incorporation of iron into proto- 

 porphyrin (Nishida and Labbe, 1959; Labbe, 1959; Minakami, 1958; 

 Krueger, Melnick and Klein, 1956; Schwartz, Cartwright, Smith and 

 Wintrobe, 1959). The information presented here substantiates the enzymic 

 nature of this important biochemical union. 



MATERIALS AND METHODS 



Approximately 200 ml of pooled blood from normal young chickens was 

 collected in a heparinized container. The blood was strained through several 

 layers of gauze and centrifuged at 1000 x g for 10 min at 4°C. The plasma 

 was discarded and the red cells washed twice with isotonic saline. Leucocytes 

 were removed by aspiration after the second washing. The cells were then 

 lysed according to the method of Dresel and Falk (1954). 



The haemolysate was centrifuged at 20,000 x ^ for 10 min at 0°C. The 

 supernatant was saved to provide carrier haem. The haemolysate residue 

 was washed twice with 0-25 m sucrose to remove haemoglobin from the 

 particulate fraction. The residue was then suspended in 0-04 m KHCO3 

 containing 10 mg/ml of Tween 40 (pH 7-4) and homogenized for 10 min in 

 a water-cooled (4°C) metal blendor cup. The suspension was allowed to settle 

 for 1 hr at 4°C and then centrifuged at 20,000 x g for 10 min. The residue 

 was discarded and mercaptoethanol (final cone. 0-01 m) added to the super- 

 natant. The bicarbonate-Tween extract was then stored at 4°C for future use. 



Protoporphyrin IX was prepared by the method of Grinstein (1947). 



* Post-doctoral Fellow of the United States Public Health Service, National Heart 

 Institute. 



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