1176 BIOCHEMISTRY: BOGORAD AND GRANICK PROC. N. A. S. I Shapley, H., and Wilson, H., Harv. Circ. 275 (1925), 276 (1925). 2 Report of the Second Conference on the Gas Dynamics of Cosmic Clouds, held at Cambridge, England, July 6-11, 1953 (in preparation). 3 These PROCEEDINGS, 39, 349-357 (1953): Harvard Reprint 372. A slightly greater value is obtained in the following paper of this series in a discussion of the bright stars in Magellanic globular clusters. 4 These PROCEEDINGS, 39, 358-362 (1953): Harvard Reprint 373. ' Henize, K. G., and F. D. Miller, Pub. Mich. Obs., 10, 75-78 (1951). 6 These PROCEEDINGS, 28, 192-199 (1942): Harvard Reprint 240. In the preceding paper of this series, these PROCEEDINGS, 39, 1161-1168 (1953), Dr. Shapley notes the evidence of some space absorption concentrated in the core. THE ENZ YMA TIC SYNTHESIS OF PORPHYRINS FROM PORPHOBILINOGEN* By LAWRENCE BOGORADt AND S. GRANICK ROCKEFELLER INSTITUTE FOR MEDICAL RESEARCH, NEW YORK Communicated by D. A. Maclnnes, October 1, 1953 Porphobilinogen has been of great interest to students of pyrrole and porphyrin metabolism since its discovery' in 1931 as a constituent of the urine of humans suffering from the hereditary disease acute porphyria. On prolonged standing at room temperatures, or much more quickly on heating, porphobilinogen gives rise to uroporphyrin with yields as high as 40%.2 The highest yields of uroporphyrin are obtained under acid condi- tions. Other as yet incompletely identified products are also formed.3 Recently porphobilinogen has been isolated and crystallized;2 structural studies4-6 indicate that it is a monopyrrole, probably of the configuration shown in figure 6. Several unsuccessful attempts to determine whether porphobilinogen may be a precursor of the naturally occurring porphyrins have been re- ported.7-9 Alternative possible relationships between porphobilinogen and pyrrole and porphyrin metabolism which have been advanced are that the compound may be a degradation product of porphyrins or that it may be some product associated with abnormal pyrrole metabolism peculiar to acute porphyria. The evidence presented in the present paper demon- strates that porphobilinogen can be used as a precursor in the biosynthesis of naturally occurring porphyrins, including protoporphyrin, by frozen and thawed preparations of Chlorella cells. Also, it has been possible to inter- rupt the enzyme system in these preparations in such a way that no detect- able amount of protoporphyrin is formed, but instead uroporphyrin I is accumulated. Other experiments are described in which enzymatic por- Downloaded by guest on September 29, 2021 VOL. 39, 1953 BIOCHEMISTRY: BOGORAD AND GRANICK 1177 phyrin synthesis from porphobilinogen has occurred in the cell-free supernate of frozen and thawed Chlorella cells. The relationship of these findings to the general problem of porphyrin biosynthesis and the porphyria diseases will be discussed. Methods and Materials.-Porphobilinogen was isolated by Westall's' method from the urine of a patient with acute porphyria. + The Chlo- rella used in these experiments were grown on a solid (agar) medium con- taining glucose and inorganic salts in diphtheria toxin bottles, each bottle containing one liter of medium. The cultures were grown at 300C. in darkness for four days and were then grown under fluorescent light for a 15 to 20-hr. period preceding harvest. The final yield of cells was about 10 cc. of packed cells per bottle. The cultures were harvested by washing the cells from the surface of the agar with distilled water. The cells were then packed by centrifugation and were frozen in the centrifuge tube in a dry ice-alcohol bath and thawed by immersing the tube in warm water. The cells were subjected to two cycles of freezing and thawing and then diluted with one-half volume of 0.25 M sucrose. Aliquots of diluted suspensions were capable of using porphobilinogen for the production of porphyrins, including protoprophyrin, with the addition of only the substrate to the suspension. However, in the experiments described in this paper other items were added as described below. Rat liver mitochondria were prepared in isotonic sucrose, essentially ac- cording to the method described by Schneider. 14 Porphobilinogen was assayed by the Ehrlich p-dimethylaminobenzalde- hyde reaction. The optical density of the pyrrole-p-dimethylamino- benzaldehyde complex was measured at 555 m,u (molar extinction for the complex = 27,400). 13 This reagent couples with free a-positions of pyr- roles and multi-pyrrolic compounds; for this reason the data given below for decrease in concentration of porphobilinogen in incubated preparations should, more strictly, be considered a decrease in number of free a-positions. Quantitative analyses of porphyrins were made spectrophotometrically using a Cary recording spectrophotometer. Qualitative analyses of por- phyrin products were made by several methods of paper chromatog- raphy. 10-13 Experiments and Results.-Growing intact normal green and mutant Chlorella cells were used in several attempts to demonstrate the utilization of porphobilinogen for the production of porphyrins, including chlorophyll. In some experiments there was a greater decrease in porphobilinogen con- centration in the presence of cells than in the controls; however, no por- phyrins other than the ones normally produced by the particular strain could be recovered. These normal products were present in such abun- dance originally that any relatively small increase in them would have been difficult to detect. Downloaded by guest on September 29, 2021 1178 BIOCHEMISTRY: BOGORAD AND GRA NICK PROC. N. A. S. Frozen and thawed preparations of normal green Chlorella prepared as described above proved very suitable for these experiments. Normal green Chlorella were used because porphyrins other than the chlorophylls, if pres- ent, occur in quantities too small to detect, particularly in the amounts of tissue required in each experiment. On the other hand it was found, as had been anticipated, that porphyrin biosynthesis from porphobilinogen, as far as could be determined, stopped short of chlorophyll, and so the new prod- ucts could be separated readily from the chlorophyll initially present. The system which was most active in synthesizing from porphobilinogen the greatest quantity of porphyrins with fewer than 8 carboxyl groups per molecule in a typical experiment contained the following components in a final volume of 4 cc.: 2 cc. of frozen and thawed diluted Chlorella suspension (prepared as described above); 0.2 cc. of rat liver mitochondria prepara- tion'4 (this volume of mitochondria suspension is approximately equal to that obtained from 1.5 g. of rat liver); adenosine triphosphate (final con- centration 5 X 10-4 M); MgCl2 (final concentration 2 X 10-3 M); and phosphate buffer pH 7.6 (final concentration 0.095 M). The pH generally fell to about pH 7 by the end of a 20-hr. incubation period under these con- ditions. Penicillin and streptomycin were found to be without effect on the system; these two compounds were added to a final concentration of 125 mg. per liter each to inhibit bacterial growth. With an initial porphobilinogen concentration of about 200 ,ug. per cc., no porphobilinogen was detectable in the suspension at the end of a 20-hr. in- cubation period at 30°C. (see below for kinetic experiments). In various experiments the new porphyrin present could account for from 50 to 70% of the porphobilinogen initially present. Controls lacking the frozen and thawed Chlorella material usually still contained about 75% of the initial porphobilinogen at the termination of the experiment, and the porphyrin formed non-enzymatically in these con- trols could account for only about 8% of the porphobilinogen that had dis- appeared. Controls containing all the components listed above but lacking porphobilinogen contained no detectable porphyrins, other than chloro- phylls and chlorophyll degFadation products, at the end of the 20-hr. in- cubation period. At the termination of the incubation period the following procedure was employed to isolate the newly formed porphyrins. The suspension (4 cc.) was centrifuged and the supernatant fluid collected. The sedimented solids were twice resuspended in water and recentrifuged, the three super- natant fractions were combined (total volume 7 cc.) and centrifuged at 14,OOOX G in the cold for 1 hr. Aliquots of the clarified supernates were taken for porphobilinogen assays, for the spectrophotometric quantitative assay, and for the qualitative analysis of porphyrins by paper chroma- tography. Downloaded by guest on September 29, 2021 VOL. 39, 1953 BIOCHEMISTRY: BOGORAD AND GRANICK 1179 In some cases these aqueous extracts were fractionated further. The extract was first washed with ether and the ether was discarded. Following this, acetic acid was added to bring the solution to about pH 4. The ether- soluble components were then extracted with ether (or in some cases ethyl acetate). Porphyrins with 2, 3, 4, 5, 6, and 7 COOH groups per molecule were detectable in such fractions by paper chromatography. Porphyrins with 4 and with 5 carboxyl groups per molecule were most abundant. The pigment which remained in the aqueous phase, usually only uroporphyrin, was concentrated by adsorption on talc; it was eluted from the talc with a small amount of concentrated NH40H. The washed sedimented material from the centrifugations described above (approximately 1 cc.) was extracted with acetone and then with acetone (10 cc.) concentrated HCI (0.1 cc.). This fraction contained water-insoluble porphyrins, including chlorophyll degradation products. The ether-soluble components of this acetone-HCl fraction were then TABLE 1 PORPHYRINS NEWLY FORMED FROM PORPHOBILINOGEN IN REACTION MIXTURE CON- TAINING FROZEN AND THAWED CHLORELLA TOTAL PORPHYRIN FRACTION PORPHYRINS PRESENTa SYNTHESIZED, % Aqueous Uroporphyrinb 28 2, 3, 4,c5, 6, 7 52 Acetone-HCl (2N HCl) 2, 4,C 5 6 Protoporphyrin 14 aNumbers refer to number of -COOH groups per porphyrin molecule, estimated on the basis of paper chromatography by Nicholas-Rimington method.10 bMostly uroporphyrin I.