208 



R. A. Neve 



One microcurie of radioactive iron was added as ^^Fe citrate to each sample. 

 The iron concentration per sample was 0-02 //m and protoporphyrin 0-05 /<m. 



Incubation of the bicarbonate-Tween extract with porphyrin and ^^Fe 

 was carried out in 50 ml Erlenmeyer flasks at 37°C for 2 hr in air on a 

 circular shaker. Labelled haemin was then isolated with carrier haemin 

 (Labbe and Nishida, 1957) and recrystallized. Sufficient haemin was dis- 

 solved to give a concentration of 2 mg/ml in 0-1 n KOH. Two ml of this 

 solution was measured for radioactivity in a Baird Atomic scintillation well 

 counter (efficiency 28 %). Results are expressed either as percentage activity 

 or counts per minute. 



RESULTS 



The optimal pH for iron incorporation into protoporphyrin was deter- 

 mined. Some workers have found a sharp peak of activity at pH 7-4 in a 



pH 



Fig. 1 . Effect of pH on enzyme activity. 



liver extract (Nishida and Labbe, 1959), while others report 7-9 as the 

 optimal pH (Schwartz et al., 1959). The results shown in Fig. 1 indicate this 

 preparation to be most active at pH 7-4. The enzyme is also extracted from 

 the nuclear fraction more easily, and is more stable during cold storage, 

 at this pH. 



The effect of the non-ionic detergent, Tween 40, is seen in Fig. 2. The 

 incorporation of iron into protoporphyrin is not dependent on the solubilizing 

 properties of the detergent during the 2 hr incubation period. In addition. 



