BIOSYNTHESIS OF UROPORPHYRIX PRECURSORS 470 



should be pointed out that the methods a^'ailable for the paper 

 chromatography of the methyl esters of the uroporphyrins aud 

 coproporphyrins (11) are not capable of distinguishing the I from 

 the II isomer types, or the III from the IV isomer types; however, 

 the melting point data support the contention that the I and the III 

 isomers are the ones being dealt with in these experiments.) These 

 data support some earlier observations (6) that frozen and thawed 

 Chlorella preparations which can normally catalyze the synthesis of a 

 number of porphyrins, including protoporphyrin IX, from PBG will 

 catalyze the synthesis of uroporphyrin I almost exclusively if main- 

 tained at 55°C. for 30 minutes prior to incubation with the substrate. 

 All these data suggest the participation of two enzymes in the syn- 

 thesis of the III isomer type as compared with the evidence that the 

 I isomer type is synthesized from PBG in the presence of the de- 

 aminase alone. 



Another ammonium sulfate fraction of aqueous extracts of wheat 

 germ is of particular interest in this connection. Preparations have 

 been obtained which do not catalyze the "consumption" or other de- 

 tectable modification of PBG; however, when such wheat germ prepa- 

 rations are incubated with PBG and PBG deaminase and then por- 

 phyrin is permitted to form, the product is approximately 85% to 90% 

 uroporphyrin III. This estimate is based on paper chromatography 

 of the uroporphyrin methyl esters, the melting point of the methyl 

 esters (crystals soften at 2o7°C.; birefringence is gone by 262°C.), 

 and the melting point of the coproporphyrin obtained by the partial 

 decarboxylation of the uroporphyrin mixture (rosettes which soften 

 at 158°C., melt at 199° to 200°C., and recrystaUize at 150° to 

 155°C.). As pointed out repeatedly above, the incubation of PBG 

 with the deaminase alone leads to a product which can be oxidized to 

 uroporphyrin I. 



This "isomerizing enzyme" from wheat germ has no effect on the 

 nature of the final porphyrin product if it is added subsequent to the 

 consumption of the PBG in the presence of the deaminase alone, 

 i.e., to a solution containing the colorless product of the reaction by 

 PBG deaminase. 



Experiments have also been performed in which PBG was in- 

 cubated with the wheat germ preparation for 3 hours, and then this 

 enzyme was inactivated by heat treatment (55°C.) ; this was fol- 

 lowed by the addition of PBG deaminase preparations from spinach. 



