254 Essays in Biochemistry 



The condensation of ''active" succinate and glycine to form 8-amino- 

 levnlinic acid subsequently thus far appears to require the partially 

 intact structure of the red blood cell. It has been found that, whereas 

 8-aminolevulinic acid can be converted to protoporphyrin in either an 

 homogenized preparation or in a cell-free extract, the conversion of 

 succinate and glycine to porphyrin takes place only with intact cells 

 or with those cells which have been hemolyzed with water. 18 Homog- 

 enized preparations obtained in a blender are no longer capable of 

 synthesizing protoporphyrin from succinate and glycine. It would 

 appear that on homogenization the functional activity of only those 

 enzymes of the system that are involved in the condensation of suc- 

 cinate and glycine is lost. However, the finding that 8-aminolevulinic 

 acid can be converted to protoporphyrin in a cell-free extract opened 

 up the possibility that soluble enzymes, concerned with each of the 

 steps in this conversion, could be isolated. 



Indeed, it was subsequently and independently found in three dif- 

 ferent laboratories that a highly purified protein fraction from ox 

 liver,- 5 duck erythrocytes, 26 and chicken erythrocytes 27 can convert 

 8-aminolevulinic acid to porphobilinogen. In our laboratory we ob- 

 tained a highly purified fraction from duck blood which on incubation 

 with 8-aminolevulinic acid-5-C 14 produced labeled porphobilinogen. 

 Since the porphobilinogen is presumably synthesized from two moles 

 of 8-aminolevulinic acid (Fig. 8), its molar radioactivity should be 

 twice that of the 8-aminolevulinic acid used as the substrate. The 

 molar radioactivities of the substrate, 8-aminolevulinic acid, and of 

 the product, porphobilinogen, were found to be 242 X 10 3 c.p.m. and 

 487 X 10 3 c.p.m. respectively. This finding demonstrates experimen- 

 tally the utilization of two moles of 8-aminolevulinic acid for porpho- 

 bilinogen formation. Further evidence that porphobilinogen is an 

 intermediate in protoporphyrin synthesis was obtained by incubating 

 equal volumes of the cell-free extract of duck erythrocytes with equi- 

 molar amounts of 8-aminolevulinic acid (0.018 mc./mM) and with the 

 enzymatically synthesized radioactive porphobilinogen (0.036 mc./mM I 

 and subsequently isolating the hemin and determining its radioactivity. 

 The radioactivities of the hemin samples synthesized from 8-amino- 

 levulinic acid and from the porphobilinogen were 92 c.p.m. and 85 

 c.p.m. respectively, after a 2-hour incubation, and 350 and 336 c.p.m. 

 respectively after a 15-hour incubation period. 20 This latter result 

 is in agreement with the findings of Falk, Dresel, and Rimington 2 " 

 and of Bogorad and Granick. 29 



