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Uroporphyrin III: Its enzymic synthesis
328
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 26784
U R O P O R P H Y R I N III: ITS ENZYMIC SYNTHESIS
HORACIO A. SANCOVICH, E L E N A B. C. LLAMB~AS, ANA MARIA FERRAMOLA, ALCIRA M. D E L C. B A T L L E A~D MOIS1~S G R I N S T E I N Cdtedra de Qulmica Biologica I, Departamento de Quimiea Bioldgica, Faeultad de Ciencias Exactas y Naturales, Ciudad Universitaria, Buenos Aires (Argentina) (Received July 23rd, 1971) (Revised manuscript received October I3th, 1971)
SUMMARY
An enzymic method has been developed for the preparation of uroporphyrin III using both porphobilinogen and porphobilinogenase from avian erythrocytes.
INTRODUCTION
Uroporphyrin III is necessary as a substrate or reference material in biochemical work. Because the natural sources or chemical methods are sometimes limited or unreliable it was desirable to work out a practical enzymic method. Uroporphyrin III is an oxidation product of the anabolic uroporphyrinogen 111. It occurs in relatively minute amounts in some normal biological materials 1-3. Considerable quantities are found in urine and tissues of patients suffering from hepatic porphyria ~,5or of animals with induced porphyria 8,7, accompanied by varying amounts of its isomer I and other porphyrins. Almost pure uroporphyrin III occurs as its copper complex in Turaco feathers 8. Urine of rats with experimental hexachlorobenzene porphyria contains relatively large quantities of uroporphyrin 1117. Pure uroporphyrin III can be obtained from these natural sources. Other good sources of this porphyrin are red cell or bovine liver preparations incubated with 6-aminolevulic acid or porphobilinogen 9-11. It can also be synthesized in the laboratory by chemical methodsl~, 13. The combined action of two enzymes, porphobilinogen deaminase (deaminase) and uroporphyrinogen III cosynthetase (isomerase), is required for the polymerization of porphobilinogen into uroporphyrinogen III (for bibliography see ref. 14). The trivial name porphobilinogenase, suggested by LOCKWOODAND RIMINGTON 15, is frequently used to designate the deaminase-isomerase complex. From avian erythrocytes a highly purified and very active porphobilinogenase 16 has been obtained. Preparations from different stages of purification of this enzyme formed mainly uroporphyrin III. Therefore, a simple procedure was developed for the enzymic preparation of uroporphyrinogen III by using both partially purified porphobilinogenase from avian erythrocytes and its substrate, porphobilinogen. Biochim. Biophys. Aeta, 261 (1972) 328-331
329
UROPORPHYRIN I I I , ITS ENZYMIC SYNTHESIS PROCEDURE
Porphobilinogen or [l*CJporphobilinogen were biosynthetically obtained 17 and assayed by the method of MOORE AND LABBExs. The enzyme used in this study, porphobilinogenase, was prepared from chicken erythrocytes by the procedure described by LLAMBfASAND BATLLE16 with some modifications. The avian blood (approx. I 1) was centrifuged, plasma and leucocytes were removed and the cells were washed with 0.9% NaC1. The packed cells were mixed thoroughly with an equal volume of water, previously chilled to o ° and periodically stirred for 60 rain in an ice-bath. The avian erythrocyte hemolysate was centrifuged at 25ooo×g for 9 ° min and the sediment discarded. The supernatant was filtered through filter paper and then placed on a column (2 c m × 5 o cm) of DEAE-cellulose previously equilibrated with 0.003 M phosphate buffer (pH 7-4)- The column was washed thoroughly with the same buffer and the DEAE-cellulose then removed mechanically. The porphyrin-synthesizing system was desorbed from the DEAE-cellulose as follows: 80-90 ml of o.134 M phosphate buffer (pH 7.4) were added to the adsorbent and the mixture was stirred magnetically for 30 rain in an ice-bath. The fluid supernatant was then collected by centrifugation at 2000 ×g for IO rain. This step was repeated three times and the supernatants were combined to yield the enzyme protein fraction. The protein solution was then fractionated with (NH,)2SO, and the fraction precipitating at 30-70% saturation was collected by centrifugation. The porphobilinogenase thus obtained was stored as a precipitate at --15°; under these conditions the enzyme is very stable for 3 months. Ammonium ions are known to inhibit porphobilinogenasel~; thus before use, the (NH,),SO,-precipitated enzyme was dissolved in a small volume of 0.05 M phosphate buffer (pH 7.4) and passed through a Sephadex G-25 column (2 cm × 3 ° cm) eluted with the same buffer. A typical incubation mixture contained: 20 ml of enzyme solution (IO mg protein per ml in 0.05 M K2HPO,-KHzPO4 buffer, pH 7.4; specific activity: I #g uroporphyrin/ mg per h), 17. 5 ml of 1.7 M NaC1, 7.5 ml of o.12 M MgC12, and 3.6 mg of porphobilinogen (or [~4C]porphobilinogen 0.o71/zC/mg), in a final volume of IOO ml adjusted with the same buffer at pH 7.4. Incubations were carried out aerobically in conical flasks in the dark with mechanical shaking at 380 for 6 h. It is known that substrate concentration and enzyme activity affect the isomer composition of the porphyrin. Care should thus be taken that the recommended substrate concentration is not exceeded and that the enzyme activity is not less than that recommended. ISOLATION AND PROPERTIES OF UROPORPHYRIN I I I
The uroporphyrin formed was isolated and purified by the following procedures: after incubation, trichloroacetic acid was added (to a final concentration of 5%, w/v) to precipitate the protein and the mixture exposed to light and air for 25 rain to oxidize the porphyrinogens. The precipitated protein was then removed by filtration and porphyrins were estimated in acid solution 21. Uroporphyrin was then isolated and esterified by the usual procedures 10. In some experiments the porphyrin was isolated in the following way: the acid solution was neutralized with sodium acetate to pH 3-o-3.5, the porphyrin was fixed on the De Acidite F F / I P resin (Permutit Co.) and then eluted with the esterification mixture, H2SO4-CH3OH, (5 %, v/v). Identification, quantitative Biochim. Biophys. Acta, 261 (1972) 328-331
330
H.A. SANCOVICHet al.
determination and determination of the isomeric type of the uroporphyrin methyl ester were made using published methods ~2-24. The uroporphyrin methyl ester was further purified by chromatography on CaC03 columns .5 and/or MgO columns 28 to obtain a chromatographic entity. The methyl ester of uroporphyrin was crystallized using methanol-chloroformt the overall yield being 55-60%. Melting points and absorption spectra were determined on the crystalline esters. Decarboxylation of uroporphyrin was carried out according to the method of EDMUNDSON AND SCHWARTZ27 and the coproporphyrin obtained was isolated and studied as detailed elsewhere 1°. The purified preparation of porphobilinogenase employed here and the incubation conditions described assure that the product of the reaction is only uroporphyrin III, occasionally accompanied by traces of phyriaporphyrin III TM, in no case higher than lO%. The uroporphyrin III methyl ester isolated by either procedure ran on paper or thin-layer chromatography as a single spot having the same RF value as the uroporphyrin III marker. A solution of the uroporphyrin III methyl ester in chloroform had absorption maxima at 626, 571, 536, 503 and 406.5 nm. The methyl ester of this biosynthetic uroporphyrin III crystallized very slowly and had a In.p. of 256-258°, identical to that of uroporphyrin III isolated from Turaco. After decarboxylation only coproporphyrin III was observed on paper or thin-layer chromatography and its methyl ester crystallized as the coproporphyrin III methyl ester, m.p. = 142° (17o°). ACKNOWLEDGEMENTS
Part of this work was supported by the Consejo Nacional de Investigaciones Cientificas y T~cnicas, Buenos Aires, Argentina. One of the authors (A. M. d. C. B.) is a member of the Career of Scientific Researchers in this Consejo. This work forms part of the thesis submitted by E.B.C.L. for the degree of Ph.D. to the University of Buenos Aires. REFERENCES 1 H. FISCHER AND H. ORTH, Die Chemie des Pyrrols, Vo]. 2, P a r t I, A k a d e m i s c h e Verlagsgesellschaft, Leipzig, 1937. 2 R. A. NEVE, R. F. LABBE AND R. A. ALDRICH, J. Am. Chem. Soc., 78 (1956) 691. 3 D. MAUZERALL AND S. GRANICK, J. Biol. Chem., 232 (1958) 141. 4 S. NACHT, L. C. SAN MARTIN DE VIALE AND M. GRINSTEIN, Clin. Chim. Acta, 27 (197 o) 4455 C. RIMINGTON, Acta Med. Scan&, 143 (1952) 177. 6 F. DE MATTEIS, B. E. PRIOR AND C. RIMINGTON, Nature, 191 (1961) 363. 7 L. C. SAN MARTIN DE VIALE, A. A. VIALE, S. NACHT AND M. GRINSTEIN, Clin. Chim. Acta, 27 (197 ° ) 13. 8 R. E. H. NICHOLAS AND C. RIMINGTON, Biochem. J., 5 ° (1951) 194. 9 J. E. FALK AND E. I. n . DRESEL, A. BENSON AND ]3. C. KNIGHT, Biochem. J., 63 (1956) 87. IO A. 1Vi. DEL C. BATLLE AND M. GRINSTEIN, Biochim. Biophys. Acta, 82 (1964) i. I i H. A. SANCOVICH, A. ~tV[.DEL C. BATLLE AND IV[. GRINSTEIN, Biochim. Biophys. Acta, 191 (1969) 13o. 12 A. TREIBS AND W. OTT, Naturwissenschaften, 4 ° (1953) 476. 13 E. J. TARLTON, S. F. MAC DONALD AND E. BALTAZZI, J. Am. Chem. Soc., 82 (196o) 4389. 14 E. B. C. LLAMBIAS AND A. 1V~.DEL C. BATLLE, Biochem. J., 121 (197 i) 327 . 15 W. LOCKWOOD AND C. RIMINGTON, Biochem. J., 67 (1957) 8 p. 16 E. B. C. LLAMBIAS AND A. M. DEL C. BATLLE, Biochim. Biophys. Acta, 227 (1971) 18o. 17 H. A. SANCOVICH, A. M. FERRAMOLA, A. M. DEL C. BATLLE AND M. GRINSTEIN, in S. P. COLOWICK AND ~ . O. KAPLAN, Methods in Enzymology, Vol. 17, A c a d e m i c Press, N e w Y o r k , 197 o, p. 220.
Biochim. Biophys. Acta, 261 (1972) 328-331
UROPORPHYRIN III, ITS ENZYMIC SYNTHESIS
331
18 D. ]. MOORE AND R. LABBE, Clin. Chem., co (1964) 11o5. 19 L. BOGORAD, Ann. N. Y. Acad. Sci., Io 4 (1963) 676. 20 O. H. LOWRY, ~T. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 21 C. RIMINGTON, Biochem. J., 15 (196o) 620. 22 A. M. DEL C. BATLLE AND M. GRINSTEIN, Abstr. 8th Latinoam. Meeting Chem., Buenos Aires, (I962), p. lO7. 23 P. CORNFORD AND A. BENSON, J. Chromatog., io (1963) 141. 24 A. M. DEL C. BATLLE AND A. BEN$ON, J. Chromatog., 25 (1966) 117. 25 M. GRINSTEIN, S. SCHWARTZ AND R. C. J. WATSON, J . Biol. Chem., 157 (1945) 323. 26 R. E. NICHOLAS AND C. RIMINGTON, Biochem. J., 48 (1951) 309. 27 P. R. EDMUNDSON AND S. SCHWAgTZ, J. Biol. Chem., 205 (1953) 605.
Biochim. Biophys. Acta, 261 (1972) 328-331
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 26784
U R O P O R P H Y R I N III: ITS ENZYMIC SYNTHESIS
HORACIO A. SANCOVICH, E L E N A B. C. LLAMB~AS, ANA MARIA FERRAMOLA, ALCIRA M. D E L C. B A T L L E A~D MOIS1~S G R I N S T E I N Cdtedra de Qulmica Biologica I, Departamento de Quimiea Bioldgica, Faeultad de Ciencias Exactas y Naturales, Ciudad Universitaria, Buenos Aires (Argentina) (Received July 23rd, 1971) (Revised manuscript received October I3th, 1971)
SUMMARY
An enzymic method has been developed for the preparation of uroporphyrin III using both porphobilinogen and porphobilinogenase from avian erythrocytes.
INTRODUCTION
Uroporphyrin III is necessary as a substrate or reference material in biochemical work. Because the natural sources or chemical methods are sometimes limited or unreliable it was desirable to work out a practical enzymic method. Uroporphyrin III is an oxidation product of the anabolic uroporphyrinogen 111. It occurs in relatively minute amounts in some normal biological materials 1-3. Considerable quantities are found in urine and tissues of patients suffering from hepatic porphyria ~,5or of animals with induced porphyria 8,7, accompanied by varying amounts of its isomer I and other porphyrins. Almost pure uroporphyrin III occurs as its copper complex in Turaco feathers 8. Urine of rats with experimental hexachlorobenzene porphyria contains relatively large quantities of uroporphyrin 1117. Pure uroporphyrin III can be obtained from these natural sources. Other good sources of this porphyrin are red cell or bovine liver preparations incubated with 6-aminolevulic acid or porphobilinogen 9-11. It can also be synthesized in the laboratory by chemical methodsl~, 13. The combined action of two enzymes, porphobilinogen deaminase (deaminase) and uroporphyrinogen III cosynthetase (isomerase), is required for the polymerization of porphobilinogen into uroporphyrinogen III (for bibliography see ref. 14). The trivial name porphobilinogenase, suggested by LOCKWOODAND RIMINGTON 15, is frequently used to designate the deaminase-isomerase complex. From avian erythrocytes a highly purified and very active porphobilinogenase 16 has been obtained. Preparations from different stages of purification of this enzyme formed mainly uroporphyrin III. Therefore, a simple procedure was developed for the enzymic preparation of uroporphyrinogen III by using both partially purified porphobilinogenase from avian erythrocytes and its substrate, porphobilinogen. Biochim. Biophys. Aeta, 261 (1972) 328-331
329
UROPORPHYRIN I I I , ITS ENZYMIC SYNTHESIS PROCEDURE
Porphobilinogen or [l*CJporphobilinogen were biosynthetically obtained 17 and assayed by the method of MOORE AND LABBExs. The enzyme used in this study, porphobilinogenase, was prepared from chicken erythrocytes by the procedure described by LLAMBfASAND BATLLE16 with some modifications. The avian blood (approx. I 1) was centrifuged, plasma and leucocytes were removed and the cells were washed with 0.9% NaC1. The packed cells were mixed thoroughly with an equal volume of water, previously chilled to o ° and periodically stirred for 60 rain in an ice-bath. The avian erythrocyte hemolysate was centrifuged at 25ooo×g for 9 ° min and the sediment discarded. The supernatant was filtered through filter paper and then placed on a column (2 c m × 5 o cm) of DEAE-cellulose previously equilibrated with 0.003 M phosphate buffer (pH 7-4)- The column was washed thoroughly with the same buffer and the DEAE-cellulose then removed mechanically. The porphyrin-synthesizing system was desorbed from the DEAE-cellulose as follows: 80-90 ml of o.134 M phosphate buffer (pH 7.4) were added to the adsorbent and the mixture was stirred magnetically for 30 rain in an ice-bath. The fluid supernatant was then collected by centrifugation at 2000 ×g for IO rain. This step was repeated three times and the supernatants were combined to yield the enzyme protein fraction. The protein solution was then fractionated with (NH,)2SO, and the fraction precipitating at 30-70% saturation was collected by centrifugation. The porphobilinogenase thus obtained was stored as a precipitate at --15°; under these conditions the enzyme is very stable for 3 months. Ammonium ions are known to inhibit porphobilinogenasel~; thus before use, the (NH,),SO,-precipitated enzyme was dissolved in a small volume of 0.05 M phosphate buffer (pH 7.4) and passed through a Sephadex G-25 column (2 cm × 3 ° cm) eluted with the same buffer. A typical incubation mixture contained: 20 ml of enzyme solution (IO mg protein per ml in 0.05 M K2HPO,-KHzPO4 buffer, pH 7.4; specific activity: I #g uroporphyrin/ mg per h), 17. 5 ml of 1.7 M NaC1, 7.5 ml of o.12 M MgC12, and 3.6 mg of porphobilinogen (or [~4C]porphobilinogen 0.o71/zC/mg), in a final volume of IOO ml adjusted with the same buffer at pH 7.4. Incubations were carried out aerobically in conical flasks in the dark with mechanical shaking at 380 for 6 h. It is known that substrate concentration and enzyme activity affect the isomer composition of the porphyrin. Care should thus be taken that the recommended substrate concentration is not exceeded and that the enzyme activity is not less than that recommended. ISOLATION AND PROPERTIES OF UROPORPHYRIN I I I
The uroporphyrin formed was isolated and purified by the following procedures: after incubation, trichloroacetic acid was added (to a final concentration of 5%, w/v) to precipitate the protein and the mixture exposed to light and air for 25 rain to oxidize the porphyrinogens. The precipitated protein was then removed by filtration and porphyrins were estimated in acid solution 21. Uroporphyrin was then isolated and esterified by the usual procedures 10. In some experiments the porphyrin was isolated in the following way: the acid solution was neutralized with sodium acetate to pH 3-o-3.5, the porphyrin was fixed on the De Acidite F F / I P resin (Permutit Co.) and then eluted with the esterification mixture, H2SO4-CH3OH, (5 %, v/v). Identification, quantitative Biochim. Biophys. Acta, 261 (1972) 328-331
330
H.A. SANCOVICHet al.
determination and determination of the isomeric type of the uroporphyrin methyl ester were made using published methods ~2-24. The uroporphyrin methyl ester was further purified by chromatography on CaC03 columns .5 and/or MgO columns 28 to obtain a chromatographic entity. The methyl ester of uroporphyrin was crystallized using methanol-chloroformt the overall yield being 55-60%. Melting points and absorption spectra were determined on the crystalline esters. Decarboxylation of uroporphyrin was carried out according to the method of EDMUNDSON AND SCHWARTZ27 and the coproporphyrin obtained was isolated and studied as detailed elsewhere 1°. The purified preparation of porphobilinogenase employed here and the incubation conditions described assure that the product of the reaction is only uroporphyrin III, occasionally accompanied by traces of phyriaporphyrin III TM, in no case higher than lO%. The uroporphyrin III methyl ester isolated by either procedure ran on paper or thin-layer chromatography as a single spot having the same RF value as the uroporphyrin III marker. A solution of the uroporphyrin III methyl ester in chloroform had absorption maxima at 626, 571, 536, 503 and 406.5 nm. The methyl ester of this biosynthetic uroporphyrin III crystallized very slowly and had a In.p. of 256-258°, identical to that of uroporphyrin III isolated from Turaco. After decarboxylation only coproporphyrin III was observed on paper or thin-layer chromatography and its methyl ester crystallized as the coproporphyrin III methyl ester, m.p. = 142° (17o°). ACKNOWLEDGEMENTS
Part of this work was supported by the Consejo Nacional de Investigaciones Cientificas y T~cnicas, Buenos Aires, Argentina. One of the authors (A. M. d. C. B.) is a member of the Career of Scientific Researchers in this Consejo. This work forms part of the thesis submitted by E.B.C.L. for the degree of Ph.D. to the University of Buenos Aires. REFERENCES 1 H. FISCHER AND H. ORTH, Die Chemie des Pyrrols, Vo]. 2, P a r t I, A k a d e m i s c h e Verlagsgesellschaft, Leipzig, 1937. 2 R. A. NEVE, R. F. LABBE AND R. A. ALDRICH, J. Am. Chem. Soc., 78 (1956) 691. 3 D. MAUZERALL AND S. GRANICK, J. Biol. Chem., 232 (1958) 141. 4 S. NACHT, L. C. SAN MARTIN DE VIALE AND M. GRINSTEIN, Clin. Chim. Acta, 27 (197 o) 4455 C. RIMINGTON, Acta Med. Scan&, 143 (1952) 177. 6 F. DE MATTEIS, B. E. PRIOR AND C. RIMINGTON, Nature, 191 (1961) 363. 7 L. C. SAN MARTIN DE VIALE, A. A. VIALE, S. NACHT AND M. GRINSTEIN, Clin. Chim. Acta, 27 (197 ° ) 13. 8 R. E. H. NICHOLAS AND C. RIMINGTON, Biochem. J., 5 ° (1951) 194. 9 J. E. FALK AND E. I. n . DRESEL, A. BENSON AND ]3. C. KNIGHT, Biochem. J., 63 (1956) 87. IO A. 1Vi. DEL C. BATLLE AND M. GRINSTEIN, Biochim. Biophys. Acta, 82 (1964) i. I i H. A. SANCOVICH, A. ~tV[.DEL C. BATLLE AND IV[. GRINSTEIN, Biochim. Biophys. Acta, 191 (1969) 13o. 12 A. TREIBS AND W. OTT, Naturwissenschaften, 4 ° (1953) 476. 13 E. J. TARLTON, S. F. MAC DONALD AND E. BALTAZZI, J. Am. Chem. Soc., 82 (196o) 4389. 14 E. B. C. LLAMBIAS AND A. 1V~.DEL C. BATLLE, Biochem. J., 121 (197 i) 327 . 15 W. LOCKWOOD AND C. RIMINGTON, Biochem. J., 67 (1957) 8 p. 16 E. B. C. LLAMBIAS AND A. M. DEL C. BATLLE, Biochim. Biophys. Acta, 227 (1971) 18o. 17 H. A. SANCOVICH, A. M. FERRAMOLA, A. M. DEL C. BATLLE AND M. GRINSTEIN, in S. P. COLOWICK AND ~ . O. KAPLAN, Methods in Enzymology, Vol. 17, A c a d e m i c Press, N e w Y o r k , 197 o, p. 220.
Biochim. Biophys. Acta, 261 (1972) 328-331
UROPORPHYRIN III, ITS ENZYMIC SYNTHESIS
331
18 D. ]. MOORE AND R. LABBE, Clin. Chem., co (1964) 11o5. 19 L. BOGORAD, Ann. N. Y. Acad. Sci., Io 4 (1963) 676. 20 O. H. LOWRY, ~T. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 21 C. RIMINGTON, Biochem. J., 15 (196o) 620. 22 A. M. DEL C. BATLLE AND M. GRINSTEIN, Abstr. 8th Latinoam. Meeting Chem., Buenos Aires, (I962), p. lO7. 23 P. CORNFORD AND A. BENSON, J. Chromatog., io (1963) 141. 24 A. M. DEL C. BATLLE AND A. BEN$ON, J. Chromatog., 25 (1966) 117. 25 M. GRINSTEIN, S. SCHWARTZ AND R. C. J. WATSON, J . Biol. Chem., 157 (1945) 323. 26 R. E. NICHOLAS AND C. RIMINGTON, Biochem. J., 48 (1951) 309. 27 P. R. EDMUNDSON AND S. SCHWAgTZ, J. Biol. Chem., 205 (1953) 605.
Biochim. Biophys. Acta, 261 (1972) 328-331