480 L. isor.ouAD 



The porphyrin ultimately produced was uroporpiiyriu isomer I, 

 i.e., there was no indication from the nature of the product that the 

 PRO had ever been exposed to the isomerizing enzyme from wheat 

 germ. The remainder of such an experiment and the nature of the 

 products are shown in Mg. 8; a tracing of a chromatogram of the 

 porphj'rin methyl esters is shown in I'^ig. 4 (12). 



Initial contents of reaction ntixlures: 



#13-#17 



0.2 ml. WG 10(40-50): 0.7 ml. 0.5 M phos. buffer pH 8.2; 1 ml. PBG 



(600 mg/ml.); 1.1 ml. H.O. 

 #18 



As above, but 0.2 ml. H.O substituted for WG 10 (40-50). 



Suhseq^ient additions and manipulations: Products: 



13. a) Incubated 180 min. at 29°C. UIII » UI 

 b) At 180 min., 0.2 ml. I17HITB added.* 



14. a) Incubated ISO min. at 29°C. 



b) Heated 30 min. at 55°C., cooled, 0.2 ml. 117HIIB 



added.* UI 



15. Incubated at 29 °C. for 20 hours. 



16. a) Heated 30 min. at 55 °C. prior to initial addition of 



PBG. Cooled, 1 ml. PBG sol. added. Incubated 180 

 min. at 29°C. 

 b) At 180 min., 0.2 ml. 117HIIB added.* UI 



17. a) In ice bath 180 min. 



b) At 180 min., 0.5> ml. 117HIIB added.* UIII »> UI 



18. o) Incubated 180 min. at 29°C. 



b) Heated 30 min. at 55°C., cooled, 0.2 ml. 1 17HIIB 



added.* UI 



* Incubated at 29°C. 17 hours. 117HIIB is a preparation from spinach-leaf 

 acetone powder which contains both PBG deaminase and the enzyme which oxi- 

 dizes the colorless precursor to lu'oporphyrin. 



Fig. 3. 



This requirement for the simultaneous presence of the deaminase, 

 of the isomerizing enzyme (obtained here from wheat germ), and of 

 PBG suggests that perhaps PBG and a polypyrrolic product of the 

 action of the first enzyme (short of a cyclized tetrapyrrole) are both 

 required as substrates for the second (isomerizing) enzyme. A hy- 

 pothesis regarding the formation of type I and type III isomers pro- 

 posed earlier by Bogorad and Granick may be pertinent (6). 



It appears, from the natiu-e of the porphyrin produced when the 



