- Email: [email protected]
[43] Preparation of stereospecifically labeled porphobilinogens
[43]
STEREOSPECIFICPORPHOB|LINOGENS
375
[43] P r e p a r a t i o n of S t e r e o s p e c i f i c a l l y Labeled Porphobilinogens
By M. AKHTAR and C. JONES The stereospecifically labeled samples of porphobilinogen (PBG) have proved to be invaluable for studying the stereochemical aspects of porphyrin (heme and chlorophyll) and corrin (vitamin B~2) biosynthesis in cell-free systems.J-4 In whole-cell systems, the PBG precursors 5-aminolevulinic acid (ALA) and succinic acid have proved more useful, as the cell membrane is not normally permeable to PBG. These syntheses of PBG have been developed in two laboratories, in Southampton and Cambridge, and specific stereochemical problems have required a range of suitably labeled substrates. The resulting syntheses are multistep and frequently complex. In practice, due to problems in chemical synthesis and the relatively ready availability of the enzyme porphobilinogen synthase (ALA dehydratase), most syntheses of labeled PBG have involved building up the molecule enzymatically 5 from ALA. This leads to complex labeling patterns as label is introduced into both halves of the PBG molecule (as it is formed from the dimerisation of ALA) (see Fig. 1). All three side chains, the acetic acid, the propionic acid, and the aminomethyl, and all four prochiral centers, C-6, C-8, C-9, and C- 11, have been stereospecifically labeled. The acetic and propionic acid side chains have generally been labeled together, whereas labeling at the C-II has also led to labeling at C-2 in the pyrrole ring. These experiments have generally included a ~4C reference label to measure rates of incorporation or to assay for loss of tritium by changes in the tritium/carbon ratio. This label will not be mentioned in the text to avoid unnecessary complexity. In most of the work performed in our laboratory, ALA itself has been built up enzymatically from succinyl-CoA and glycine (Fig. 2). This chapter will deal first with the use of labeled succinic acid derivatives, which introduce label into the acetate and propionate side chains of PBG, and then with the use of labeled glycine, which is incorporated into t M. M. A b b o u d , P. M. Jordan, and M. Akhtar, Nouv. J. Chim. 2, 419 (1978). 2 A. R. Battersby, A. L. G u t m a n , C. J. R. Fookes, H. Gunther, and H. Simon, J. Chem. Soc., Chem. Commun. p. 645 (1981). 3 C. Jones, P. M. Jordan, and M. Akhtar, J. Chem. Soc.. Perkin Trans. / p. 2625 (1984). 4 j. S. Seehra, P. M. Jordan, and M. Akhtar, Biochem. J. 209, 709 (1983). 5 H. A. Sancovich, A. M. Ferramola, A. M. del C. Battle, and M. Grinstein, this series, Vol. 17, Part A, p. 220.
METHODS IN ENZYMOLOGY, VOL. 123
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
376
HEME PORPHYRINSAND DERIVATIVES
COOH ..'HR, 9r,,~ Hsi Hsi~
COOH ]
[43]
COOH
"'1%<
'~N,,~ NH2 , NH 2 H ,/ ~ O H~ Hs~ 2 FIG. 1. ALA dehydratase-catalyzedformationof porphobilinogen(PBG, 2). the aminomethyl side chain of PBG. For convenience, prototype methods will be described in detail and possible variations considered briefly. Labeling the Acid Side Chains of PBG Using Enzymatic Approaches Labeling of the acid side chains was achieved biosynthetically with the stereospecific label originating from succinic acid. The labeled succinic acid was prepared in turn by decarboxylation of a stereospecifically labeled 2-oxoglutarate, prepared by essentially literature methods. 6 As these syntheses pass through a succinic acid intermediate, symmetrical apart from the isotopic labeling, the final labeling is distributed equally between C-2 and C-3 of the ALA, and hence between C-6, C-8, and C-9 of the PBG (Fig. 3).
Preparation of [6R,8R,9R-SHs]PBG (2a) 1.4 Reagents Disodium 2-oxoglutarate Tritiated water Triethanolamine-HCl, pH 7.4 buffer, 0.5 M Isocitrate dehydrogenase (from pig heart, Sigma) NADPH, 6 mM in distilled water (pH to 7 if necessary) MgCI2.6H20, 6 mM in distilled water Hydrogen peroxide, 5% Dowex chloride l-X2 (mesh size 50-100, Sigma) Dowex chloride l-X8 (mesh size 20-50, BDH) HCI, 0.1 M in water 6G. E. Lienhardand I. A. Rose, Biochemistry3, 185 (1964).
[43]
STEREOSPECIFIC PORPHOBILINOGENS
377
COOH o 2
Hoo~ANH2
H2
Flo. 2. ALA synthetase-catalyzed formation of ALA. Disodium 2-oxoglutarate (100 mg = 526/zmol) was dissolved in tritiated w a t e r (5 Ci/ml: 1 ml) and sealed in uacuo. The tube was autoclaved at 110 ° for 10 min, cooled, opened, and the tritiated disodium 2-oxoglutarate was isolated by repeated lyophilization. The specific activity of the 2oxoglutarate was typically 20-25/xCi//~moi. D i s o d i u m 2-Oxo[3R--~Hl]glutarate a n d [ 2 R J H l ] S u c c i n a t e
Disodium 2-oxo[3RS-3Hz]glutarate (60/.~mol; 1.42 mCi) was dissolved in a solution p r e p a r e d f r o m 0.5 M t r i e t h a n o l a m i n e - H C l buffer, p H 7.4 (2 ml), N A D P H (1 ml), m a g n e s i u m chloride (I ml), and isocitrate dehydrogenase (25 units) m a d e to 10 ml with water. Samples (0.1 ml) were rem o v e d at timed intervals and applied onto a column of D o w e x chloride (IX2, 100 mesh; 0.5 × 4 cm) that was washed with w a t e r (I0 ml), and the radioactivity in the eluent was m e a s u r e d to determine tritium released in the medium. After 50% of the tritium in the 2-oxo[3-3H2]glutarate had been released into the medium, the reaction was stopped by the addition
COOH ~,,~Hsi COOH Hs~ "'HR, J~Hs, H~'eXXff '- ~ ""HR, H FIG. 3. PBG containing isotopic hydrogen atoms at C-6, C-8, and C-9. As described: [6R,8R,9R-3H31PBG(2a); 6HR~,8HRe,and 9HRe = 3H. Variation A: [6S,8S,9S-3Ha]PBG(2b); 6Hsi, 8Hsi, and 9Hsi = 3H. Variation B: (6R,8R,9R)-[6,8,9-3H2, 6,8,9, ;H3]PBG(2¢);all HR~= 3H and Hsi = 2H.
378
HEME PORPHYRINS AND DERIVATIVES
[43]
of 5% hydrogen peroxide (6.6 ml). The [2R-3Hl]succinate was purified by ion-exchange chromatography on a column of Dowex chloride (I-X8, 2050 mesh; 2.5 × 10 cm). After washing the column with water (50 ml) the succinic acid was eluted with 0.1 M hydrochloric acid (approximately 20 ml) and recovered by evaporation under reduced pressure. Yield was 50 /zmol (85%), specific radioactivity 11.84 /zCi//zmoi. This material was diluted with carrier succinic acid to give specific radioactivity as desired.
[2R,3R J H2]Succinyl-CoA Reagents Dicyclohexylcarbodiimide Acetone (dry; distilled over K2CO3) Diethyl ether (freshly dried; distilled over LiA1H4) Coenzyme A (trilithium salt) NaHCO3 The labeled succinic acid (200/zmol) obtained above was dissolved in anhydrous acetone (5 ml) and dicyclohexylcarbodiimide (200 /zmol) added. The reaction was stirred at room temperature for 30 min and further carbodiimide (20/xmol) added. After a further 30 min the dicyclohexylurea was removed by filtration and the acetone removed under reduced pressure. The succinic anhydride was extracted from the residue with dry ether (3 × 10 ml) and the ether removed under reduced pressure to give the succinic anhydride (160-170/zmol). Succinyl-CoA was prepared immediately prior to use by dissolving the succinic anhydride (90/zmol) in a solution of sodium bicarbonate (4.5 mg) and lithium coenzyme A (10 mg) in 2.5 ml of water. 7 The solution was stirred at 0° for 30 min. Complete reaction of the thiol was confirmed by treatment of 5/zl of the solution with 500/xl of a 1 mM solution of 5,5'dithiobis(2-nitrobenzoic acid) (for details, see Ref. 8).
[6R,8R,9R-3H3]PBG (2a; Fig. 3) Reagents Glycine, 400 mM in distilled water Tris-HC1 buffer, pH 6.8, 50 mM Pyridoxal phosphate 1 mM in distilled water A L A synthetase from R. spheroides 8 ALA dehydratase from bovine liver9 7 E. J. Simon and D. Shemin, J. A m . Chem. Soc. 757 2520 (1953). 8 p. M. Jordan and A. L a g h a i - N e w t o n , this volume [52].
9 p. M. Jordan and J. S. Seehra, this volume [51].
[43]
STEREOSPECIFICPORPHOBILINOGENS
379
The following stock solutions were mixed: glycine (2 ml), buffer (1 ml), pyridoxal phosphate (1 ml), ALA synthetase 8 (200 units), and ALA dehydratase 9 (350 units) and the solution made up to 7.5 ml with distilled water. To this was added the succinyl-CoA solution (2.5 ml, see above) and the mixture incubated at 37° under nitrogen until PBG formation was complete (usually 30-60 min) as judged by the reaction with p-dimethylaminobenzaldehyde.~° The mixture was adjusted to pH 7.2 and poured onto a column of Dowex acetate (2-X8, 400 mesh; 2 x 10 cm) and the resin washed with distilled water (40 ml) to remove ALA. The washings were checked for the absence of PBG. The PBG was eluted with ice cold I M acetic acid (20 ml), the solvent removed by rotary evaporation under high vacuum below 30° and the PBG further purified by thin layer chromatography on microcrystalline cellulose plates (1 mm thick, 20 x 10 cm), developed in l-butanol : acetic acid : water, 63 : 27 : 10. The PBG was located by radioactive scanning or by spraying a portion of the plate with the modified Ehrlich reagent/° and the band eluted with dilute aqueous ammonia and lyophilized. The sample of [6R,8R,9R-3H3]PBG was stored desiccated at - 18°.
Crystallization of PBG PBG was dissolved in 0.5 M aqueous ammonia (0.2 ml) and crystallized by addition of 0.5 M aqueous acetic acid until the pH was 5.0. The mixture was stood on ice for 3 hr. The crystals were removed by centrifugation, washed with methanol and dried under high vacuum.
Variations [6S,8S,gS-~H3]PBG (2b, Fig. 3). The use of unlabeled 2-oxoglutarate and an isocitrate dehydrogenase enzyme system in tritiated water (typically 2.5 Ci/ml) in a sealed tube leads to preparation of the [2S-3Hz]succinate, and hence [6S,8S,9S-3H3]PBG. 4 2-Oxoglutarate (68.5 /zmol) was dissolved in triethanolamine/HC! buffer pH 7.4 (0.5 M; 0.2 ml) containing NADPH (30 mM) and isocitrate dehydrogenase (12 units; 0.8 ml). Tritiated water (1 ml; 5 Ci) was added and the tube sealed and incubated at 30° for 3 hr. Reaction was terminated by addition of hydrogen peroxide (1.5 ml: 30%) and after 30 min the succinate was purified as before on a column of Dowex chloride (I-X8, 20-50 mesh) and used for the preparation of PBG. In this preparation the adsorption of succinate on Dowex is performed in a well-ventilated fume cupboard and the washings are collected carefully but rapidly. The recov"~D. Mauzerall and S. Granick. J. Biol. Chem. 219, 435 (1956).
380
HEME PORPHYRINS AND DERIVATIVES
[43]
ered tritiated washings are either stored for disposal or used for recovery of tritiated water by freeze drying. [6R,8R,9R-~t13, 6S,8S,9S-2H3]PBG (2a, Fig. 3). The use of 2-oxo[3RS-3Hz]glutarate and an isocitrate dehydrogenase enzyme system exchanged into deuterated water leads to the preparation of the deuteratedtritiated species, [2R-3H1, 2S-2Hl]succinatefl ~ which was used for the preparation of [6R,8R,9R-3H3, 6S,8S,9S-2H3]PBG. 12 Ethanolamine/HCl buffer, pH 7.4 (0.5 M; 10 ml) was freeze dried and redissolved in deuterated water (99% D), refreeze dried, and again made up in deuterated water. No allowances were made for the differences in pH and pD. Isocitrate dehydrogenase (50 units, 1 ml) was dialyzed against 3 × 10 ml of deuterated water at 4 ° for a total of 6 hr, in a sealed tube. The enzyme was used immediately as its stability under these conditions was suspect. The equilibration was run in the presence of 0.6 mM MgC12 and 0.6 mM NADPH as described above (stoppered under nitrogen) with monitoring of the labilization of tritium. Succinate was isolated and incorporated into [ 6 S , 8 S , 9 S - 3 H 3 , 6R,8R,9R-2H3]PBG essentially as described above. The bacterial ALA dehydratase from R. spheroides can also be used in PBG synthesis in a two-step procedure with isolation of the intermediate ALA. 1 The failure of the above method to allow regioselective labeling was solved by the Cambridge and Munich groups, which synthesized2 a stereospecifically labeled, succinic acid derivative monomethyl[2S-3Hj]succinate, which was then converted to ALA by known reactions (see Fig. 4). In principle, this later species may be converted to PBG using ALA dehydratase. The Cambridge group has also described a method for labeling the propionate side chains in which the relative, rather than absolute, stereochemistry at C-8 and C-9 is controlled. 13 Full experimental details of these syntheses are not yet available. 2,~3 A m i n o m e t h y l Side C h a i n L a b e l i n g 3 D u r i n g the c o u r s e o f o u r s t u d i e s o n the m e c h a n i s m o f a c t i o n o f A L A s y n t h e t a s e , it was s h o w n 14 that the label f r o m [2-3H2]glycine is s t e r e o s p e cifically i n c o r p o r a t e d into C-5 o f A L A (Fig. 2, in s t r u c t u r e 1 Hsi = 3H). " G. F. Barnard and M. Akhtar, J. Chem. Soc., Perkin Trans. / p. 2354 (1979). 12C. Jones and M. Akhtar, unpublished experiments (1977). 13A. R. Battersby, E. McDonald, H. K. W. Wurziger, and K. J. James, J. Chem. Soc., Chem. Commun. p. 493 (1979). 14M. M. Abboud, P. M. Jordan, and M. Akhtar, J. Chem. Soc., Chem. Commun. p. 643 (1974).
[43]
STEREOSPECIFIC PORPHOBILINOGENS
381
2H zH 3HTCOOMe > 3H-y COOMe >3H_yCOOH HOOC /
HOOC ,jI'-2H
'H~ - ~ O ~NH 2
Fxo. 4. Stereospecific introduction of hydrogen atoms at C-2 of ALA. In the first step, stereospecific reduction of the tritiated substrate in ZH20 using 2-enoate reductase gives methyl succinate which is then converted to (2S)-[2-3H,2-ZH]ALAchemically.
The stereospecifically labeled ALA thus formed may be trapped to give PBG stereospecifically labeled in the aminomethyl sidechain. 3.~5The preparative procedure is essentially similar to the coupled e n z y m e system described above, except that incubation conditions were chosen 3 to maximize the radiochemical incorporation of glycine rather than succinyl CoA, by reducing the glycine concentration from about 130 to 3 mM (Kin about 12 raM), and the reaction was performed in phosphate buffer rather than the Tris buffer. The complications arising from the nonenzymatic racemisation of the chiral center at C-5 of ALA catalyzed by pyridoxal phosphate (required as cofactor by the A L A synthetase) were avoided by using as low a concentration of pyridoxal phosphate as possible. Furthermore the system was loaded with ALA dehydratase to reduce the life of A L A in solution. Careful control of the thiol concentration was also felt to be important (for further details, see Ref. 16). It should be noted that this method has only ever been carried out on a small scale and is not suitable for deuterium labeling (Fig. 5). Reagents
[2RS-3Hz]Glycine (3/~mol, freeze-dried powder) Pyridoxal phosphate, 60/~M in distilled water T r i s - H C l buffer, 10 mM Potassium phosphate buffer, pH 6.8, l0 mM prepared in glycerol : water (2 : 8) which was also 2 m M in 2-mercaptoethanol Potassium phosphate buffer, pH 6.8, 10 m M which was also 20 mM in 2-mercaptoethanol Succinyl-CoA (3.75/~mol in 150 ~1 prepared as in Ref. 8) A L A synthetase and A L A dehydratase Dowex acetate, 2-X8 (400 mesh) 15C. Jones, P. M. Jordan, A. G. Chaudhry, and M. Akhtar, J. Chem. Soc., Chem. Commun. p. 96 (1979). ~6For further details, see C. Jones, Ph.D. Thesis, University of Southampton (1979).
382
HEME PORPHYRINS AND DERIVATIVES
[43]
COOH COOH
HR~ Hsi Fro. 5. PBG stereospeciflcal]y labeled at the aminomethyl group.
ALA synthetase and ALA dehydratase were prepared as described in Refs. 8 and 9. Succinyl-CoA was prepared from unlabeled succinic anhydride as described in Ref. 8. ALA synthetase 9 was dialyzed for 3 hr against 100 ml of the potassium phosphate buffer, pH 6.8 (10 mM) containing glycerol (20%) and 2-mercaptoethanol (2 mM) before use. ALA dehydratase8 was dissolved in a small volume of 10 mM potassium phosphate buffer containing 20 mM 2-mercaptoethanol and dialyzed against this buffer for 3 hr prior to use. The incubation mixture was prepared from [2-3H2]glycine (3 /xmol), pyridoxal phosphate (0.1 ml), Tris-HC1 buffer (0.1 ml), ALA synthetase (20 units), and ALA dehydratase (20 units) made up to 1 ml with distilled water. The final phosphate and thiol concentrations were estimated at 8 and 7 mM, respectively. The solution was warmed to 37° and reaction started by the addition of succinyl-CoA (1.25 ~mol in 50/xl; for preparation see above): further aliquots were added after 10 and 20 min. After 30 min reaction was stopped by applying it to a column of Dowex acetate (2 x 8,400 mesh; 0.5 x 6 cm) and the PBG isolated and further purified by TLC as above. The PBG from the ion exchange column was typically 98% radiochemically pure (impurities were ALA and glycine) and could often be used without further purification. Isolated radiochemical yield of the [2,11S-3H2]PBG was typically 30%. The enantiomeric excess of the product was about 84% as assayed using the protocol described in Ref. 3 (also see Ref. 17).
Variations [IIS-3HI]PBG. Dilute acid treatment of the PBG leads to exchange of the H-2 tritium in the doubly labeled PBG above. The labeled PBG (1 mg) 17 L. Schirch, this series, Vol. 17, Part B, p. 335; P. M. Jordan and M. Akhtar, Biochem. J. 116, 277 (1970).
[44]
HPLC
OF URO- AND COPROPORPHYRIN
ISOMERS
383
was dissolved in 0.15 M hydrochloric acid (100/A) and allowed to stand at room temperature for 1 hr, freeze dried, and the [11S-3H1]PBG purified by T L C on cellulose. Acknowledgments We thank SERC for their support of our work in the area of porphyrin biosynthesis.
[44] H i g h - P e r f o r m a n c e Uroporphyrin
Liquid Chromatography
and Coproporphyrin
of
Isomers
B y C. K. LIM and T. J. PETERS
Uroporphyrinogen III, the universal precursor of heme, chlorophylls, and vitamin Bt2, is derived from porphobilinogen (PBG) by the sequential action of porphobilinogen deaminase (hydroxymethylbilane synthase, EC 4.3.1.8) which catalyzes the formation of hydroxymethylbilane (HMB) from four molecules of PBG and uroporphyrinogen III synthase (EC 4.2.1.75) which converts HMB into uroporphyrinogen IlI. j A small amount uroporphyrinogen I is also formed by the spontaneous chemical ring-closure of HMB. The stepwise decarboxylation of the acetic acid groups of uroporphyrinogens I and III form the corresponding coproporphyrinogen isomers 2 and is catalyzed by uroporphyrinogen decarboxylase (EC 4.1.I.37). Porphyrins are formed by oxidation of the porphyrinogens. High-performance liquid chromatography (HPLC) has been shown to be the most effective technique for the separation of uro- and coproporphyrin isomers 3 (Fig. 1). The methyl esters of the isomers can be resolved on silica 4-6 and aminopropyl silica 7 or the porphyrin free acids can be
A. R. Battersby, C. J. R. Fookes, K. E. Gustafson-Potter, E. McDonald, and G. W. J. Matcham, J. Chem. Soc., Perkin Trans. l, p. 2427 (1982). 2 A. H. Jackson, H. A. Sancovich, A. M. Ferramola, N. Evans, D. E. Games, S. A. Matlin, G. H. Elder, and S. G. Smith, Philos. Trans. R. Sot'. London, Ser. B 273, 191 (1976). 3 C. K. Lim, J. M. Rideout, and D. J. Wright, J. Chromatogr. 282, 629 (1983). 4 H. Nordlov, P. M. Jordan, G. Burton, and A. I. Scot, J. Chromatogr. 190, 221 (1980). 5 A. H. Jackson, K. R. N. Rao, and S. G. Smith, Biochem. J. 203, 515 (1982). 6 A. H. Jackson, K. R. N. Rao, and S. G. Smith, Biochem. J. 207, 599 (1982). 7 I. C. Walker, M. T. Gilbert, and K. Stubbs, J. Chromatogr. 202, 491 (1980).
METHODS IN ENZYMOLOGY, VOL. 123
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
STEREOSPECIFICPORPHOB|LINOGENS
375
[43] P r e p a r a t i o n of S t e r e o s p e c i f i c a l l y Labeled Porphobilinogens
By M. AKHTAR and C. JONES The stereospecifically labeled samples of porphobilinogen (PBG) have proved to be invaluable for studying the stereochemical aspects of porphyrin (heme and chlorophyll) and corrin (vitamin B~2) biosynthesis in cell-free systems.J-4 In whole-cell systems, the PBG precursors 5-aminolevulinic acid (ALA) and succinic acid have proved more useful, as the cell membrane is not normally permeable to PBG. These syntheses of PBG have been developed in two laboratories, in Southampton and Cambridge, and specific stereochemical problems have required a range of suitably labeled substrates. The resulting syntheses are multistep and frequently complex. In practice, due to problems in chemical synthesis and the relatively ready availability of the enzyme porphobilinogen synthase (ALA dehydratase), most syntheses of labeled PBG have involved building up the molecule enzymatically 5 from ALA. This leads to complex labeling patterns as label is introduced into both halves of the PBG molecule (as it is formed from the dimerisation of ALA) (see Fig. 1). All three side chains, the acetic acid, the propionic acid, and the aminomethyl, and all four prochiral centers, C-6, C-8, C-9, and C- 11, have been stereospecifically labeled. The acetic and propionic acid side chains have generally been labeled together, whereas labeling at the C-II has also led to labeling at C-2 in the pyrrole ring. These experiments have generally included a ~4C reference label to measure rates of incorporation or to assay for loss of tritium by changes in the tritium/carbon ratio. This label will not be mentioned in the text to avoid unnecessary complexity. In most of the work performed in our laboratory, ALA itself has been built up enzymatically from succinyl-CoA and glycine (Fig. 2). This chapter will deal first with the use of labeled succinic acid derivatives, which introduce label into the acetate and propionate side chains of PBG, and then with the use of labeled glycine, which is incorporated into t M. M. A b b o u d , P. M. Jordan, and M. Akhtar, Nouv. J. Chim. 2, 419 (1978). 2 A. R. Battersby, A. L. G u t m a n , C. J. R. Fookes, H. Gunther, and H. Simon, J. Chem. Soc., Chem. Commun. p. 645 (1981). 3 C. Jones, P. M. Jordan, and M. Akhtar, J. Chem. Soc.. Perkin Trans. / p. 2625 (1984). 4 j. S. Seehra, P. M. Jordan, and M. Akhtar, Biochem. J. 209, 709 (1983). 5 H. A. Sancovich, A. M. Ferramola, A. M. del C. Battle, and M. Grinstein, this series, Vol. 17, Part A, p. 220.
METHODS IN ENZYMOLOGY, VOL. 123
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
376
HEME PORPHYRINSAND DERIVATIVES
COOH ..'HR, 9r,,~ Hsi Hsi~
COOH ]
[43]
COOH
"'1%<
'~N,,~ NH2 , NH 2 H ,/ ~ O H~ Hs~ 2 FIG. 1. ALA dehydratase-catalyzedformationof porphobilinogen(PBG, 2). the aminomethyl side chain of PBG. For convenience, prototype methods will be described in detail and possible variations considered briefly. Labeling the Acid Side Chains of PBG Using Enzymatic Approaches Labeling of the acid side chains was achieved biosynthetically with the stereospecific label originating from succinic acid. The labeled succinic acid was prepared in turn by decarboxylation of a stereospecifically labeled 2-oxoglutarate, prepared by essentially literature methods. 6 As these syntheses pass through a succinic acid intermediate, symmetrical apart from the isotopic labeling, the final labeling is distributed equally between C-2 and C-3 of the ALA, and hence between C-6, C-8, and C-9 of the PBG (Fig. 3).
Preparation of [6R,8R,9R-SHs]PBG (2a) 1.4 Reagents Disodium 2-oxoglutarate Tritiated water Triethanolamine-HCl, pH 7.4 buffer, 0.5 M Isocitrate dehydrogenase (from pig heart, Sigma) NADPH, 6 mM in distilled water (pH to 7 if necessary) MgCI2.6H20, 6 mM in distilled water Hydrogen peroxide, 5% Dowex chloride l-X2 (mesh size 50-100, Sigma) Dowex chloride l-X8 (mesh size 20-50, BDH) HCI, 0.1 M in water 6G. E. Lienhardand I. A. Rose, Biochemistry3, 185 (1964).
[43]
STEREOSPECIFIC PORPHOBILINOGENS
377
COOH o 2
Hoo~ANH2
H2
Flo. 2. ALA synthetase-catalyzed formation of ALA. Disodium 2-oxoglutarate (100 mg = 526/zmol) was dissolved in tritiated w a t e r (5 Ci/ml: 1 ml) and sealed in uacuo. The tube was autoclaved at 110 ° for 10 min, cooled, opened, and the tritiated disodium 2-oxoglutarate was isolated by repeated lyophilization. The specific activity of the 2oxoglutarate was typically 20-25/xCi//~moi. D i s o d i u m 2-Oxo[3R--~Hl]glutarate a n d [ 2 R J H l ] S u c c i n a t e
Disodium 2-oxo[3RS-3Hz]glutarate (60/.~mol; 1.42 mCi) was dissolved in a solution p r e p a r e d f r o m 0.5 M t r i e t h a n o l a m i n e - H C l buffer, p H 7.4 (2 ml), N A D P H (1 ml), m a g n e s i u m chloride (I ml), and isocitrate dehydrogenase (25 units) m a d e to 10 ml with water. Samples (0.1 ml) were rem o v e d at timed intervals and applied onto a column of D o w e x chloride (IX2, 100 mesh; 0.5 × 4 cm) that was washed with w a t e r (I0 ml), and the radioactivity in the eluent was m e a s u r e d to determine tritium released in the medium. After 50% of the tritium in the 2-oxo[3-3H2]glutarate had been released into the medium, the reaction was stopped by the addition
COOH ~,,~Hsi COOH Hs~ "'HR, J~Hs, H~'eXXff '- ~ ""HR, H FIG. 3. PBG containing isotopic hydrogen atoms at C-6, C-8, and C-9. As described: [6R,8R,9R-3H31PBG(2a); 6HR~,8HRe,and 9HRe = 3H. Variation A: [6S,8S,9S-3Ha]PBG(2b); 6Hsi, 8Hsi, and 9Hsi = 3H. Variation B: (6R,8R,9R)-[6,8,9-3H2, 6,8,9, ;H3]PBG(2¢);all HR~= 3H and Hsi = 2H.
378
HEME PORPHYRINS AND DERIVATIVES
[43]
of 5% hydrogen peroxide (6.6 ml). The [2R-3Hl]succinate was purified by ion-exchange chromatography on a column of Dowex chloride (I-X8, 2050 mesh; 2.5 × 10 cm). After washing the column with water (50 ml) the succinic acid was eluted with 0.1 M hydrochloric acid (approximately 20 ml) and recovered by evaporation under reduced pressure. Yield was 50 /zmol (85%), specific radioactivity 11.84 /zCi//zmoi. This material was diluted with carrier succinic acid to give specific radioactivity as desired.
[2R,3R J H2]Succinyl-CoA Reagents Dicyclohexylcarbodiimide Acetone (dry; distilled over K2CO3) Diethyl ether (freshly dried; distilled over LiA1H4) Coenzyme A (trilithium salt) NaHCO3 The labeled succinic acid (200/zmol) obtained above was dissolved in anhydrous acetone (5 ml) and dicyclohexylcarbodiimide (200 /zmol) added. The reaction was stirred at room temperature for 30 min and further carbodiimide (20/xmol) added. After a further 30 min the dicyclohexylurea was removed by filtration and the acetone removed under reduced pressure. The succinic anhydride was extracted from the residue with dry ether (3 × 10 ml) and the ether removed under reduced pressure to give the succinic anhydride (160-170/zmol). Succinyl-CoA was prepared immediately prior to use by dissolving the succinic anhydride (90/zmol) in a solution of sodium bicarbonate (4.5 mg) and lithium coenzyme A (10 mg) in 2.5 ml of water. 7 The solution was stirred at 0° for 30 min. Complete reaction of the thiol was confirmed by treatment of 5/zl of the solution with 500/xl of a 1 mM solution of 5,5'dithiobis(2-nitrobenzoic acid) (for details, see Ref. 8).
[6R,8R,9R-3H3]PBG (2a; Fig. 3) Reagents Glycine, 400 mM in distilled water Tris-HC1 buffer, pH 6.8, 50 mM Pyridoxal phosphate 1 mM in distilled water A L A synthetase from R. spheroides 8 ALA dehydratase from bovine liver9 7 E. J. Simon and D. Shemin, J. A m . Chem. Soc. 757 2520 (1953). 8 p. M. Jordan and A. L a g h a i - N e w t o n , this volume [52].
9 p. M. Jordan and J. S. Seehra, this volume [51].
[43]
STEREOSPECIFICPORPHOBILINOGENS
379
The following stock solutions were mixed: glycine (2 ml), buffer (1 ml), pyridoxal phosphate (1 ml), ALA synthetase 8 (200 units), and ALA dehydratase 9 (350 units) and the solution made up to 7.5 ml with distilled water. To this was added the succinyl-CoA solution (2.5 ml, see above) and the mixture incubated at 37° under nitrogen until PBG formation was complete (usually 30-60 min) as judged by the reaction with p-dimethylaminobenzaldehyde.~° The mixture was adjusted to pH 7.2 and poured onto a column of Dowex acetate (2-X8, 400 mesh; 2 x 10 cm) and the resin washed with distilled water (40 ml) to remove ALA. The washings were checked for the absence of PBG. The PBG was eluted with ice cold I M acetic acid (20 ml), the solvent removed by rotary evaporation under high vacuum below 30° and the PBG further purified by thin layer chromatography on microcrystalline cellulose plates (1 mm thick, 20 x 10 cm), developed in l-butanol : acetic acid : water, 63 : 27 : 10. The PBG was located by radioactive scanning or by spraying a portion of the plate with the modified Ehrlich reagent/° and the band eluted with dilute aqueous ammonia and lyophilized. The sample of [6R,8R,9R-3H3]PBG was stored desiccated at - 18°.
Crystallization of PBG PBG was dissolved in 0.5 M aqueous ammonia (0.2 ml) and crystallized by addition of 0.5 M aqueous acetic acid until the pH was 5.0. The mixture was stood on ice for 3 hr. The crystals were removed by centrifugation, washed with methanol and dried under high vacuum.
Variations [6S,8S,gS-~H3]PBG (2b, Fig. 3). The use of unlabeled 2-oxoglutarate and an isocitrate dehydrogenase enzyme system in tritiated water (typically 2.5 Ci/ml) in a sealed tube leads to preparation of the [2S-3Hz]succinate, and hence [6S,8S,9S-3H3]PBG. 4 2-Oxoglutarate (68.5 /zmol) was dissolved in triethanolamine/HC! buffer pH 7.4 (0.5 M; 0.2 ml) containing NADPH (30 mM) and isocitrate dehydrogenase (12 units; 0.8 ml). Tritiated water (1 ml; 5 Ci) was added and the tube sealed and incubated at 30° for 3 hr. Reaction was terminated by addition of hydrogen peroxide (1.5 ml: 30%) and after 30 min the succinate was purified as before on a column of Dowex chloride (I-X8, 20-50 mesh) and used for the preparation of PBG. In this preparation the adsorption of succinate on Dowex is performed in a well-ventilated fume cupboard and the washings are collected carefully but rapidly. The recov"~D. Mauzerall and S. Granick. J. Biol. Chem. 219, 435 (1956).
380
HEME PORPHYRINS AND DERIVATIVES
[43]
ered tritiated washings are either stored for disposal or used for recovery of tritiated water by freeze drying. [6R,8R,9R-~t13, 6S,8S,9S-2H3]PBG (2a, Fig. 3). The use of 2-oxo[3RS-3Hz]glutarate and an isocitrate dehydrogenase enzyme system exchanged into deuterated water leads to the preparation of the deuteratedtritiated species, [2R-3H1, 2S-2Hl]succinatefl ~ which was used for the preparation of [6R,8R,9R-3H3, 6S,8S,9S-2H3]PBG. 12 Ethanolamine/HCl buffer, pH 7.4 (0.5 M; 10 ml) was freeze dried and redissolved in deuterated water (99% D), refreeze dried, and again made up in deuterated water. No allowances were made for the differences in pH and pD. Isocitrate dehydrogenase (50 units, 1 ml) was dialyzed against 3 × 10 ml of deuterated water at 4 ° for a total of 6 hr, in a sealed tube. The enzyme was used immediately as its stability under these conditions was suspect. The equilibration was run in the presence of 0.6 mM MgC12 and 0.6 mM NADPH as described above (stoppered under nitrogen) with monitoring of the labilization of tritium. Succinate was isolated and incorporated into [ 6 S , 8 S , 9 S - 3 H 3 , 6R,8R,9R-2H3]PBG essentially as described above. The bacterial ALA dehydratase from R. spheroides can also be used in PBG synthesis in a two-step procedure with isolation of the intermediate ALA. 1 The failure of the above method to allow regioselective labeling was solved by the Cambridge and Munich groups, which synthesized2 a stereospecifically labeled, succinic acid derivative monomethyl[2S-3Hj]succinate, which was then converted to ALA by known reactions (see Fig. 4). In principle, this later species may be converted to PBG using ALA dehydratase. The Cambridge group has also described a method for labeling the propionate side chains in which the relative, rather than absolute, stereochemistry at C-8 and C-9 is controlled. 13 Full experimental details of these syntheses are not yet available. 2,~3 A m i n o m e t h y l Side C h a i n L a b e l i n g 3 D u r i n g the c o u r s e o f o u r s t u d i e s o n the m e c h a n i s m o f a c t i o n o f A L A s y n t h e t a s e , it was s h o w n 14 that the label f r o m [2-3H2]glycine is s t e r e o s p e cifically i n c o r p o r a t e d into C-5 o f A L A (Fig. 2, in s t r u c t u r e 1 Hsi = 3H). " G. F. Barnard and M. Akhtar, J. Chem. Soc., Perkin Trans. / p. 2354 (1979). 12C. Jones and M. Akhtar, unpublished experiments (1977). 13A. R. Battersby, E. McDonald, H. K. W. Wurziger, and K. J. James, J. Chem. Soc., Chem. Commun. p. 493 (1979). 14M. M. Abboud, P. M. Jordan, and M. Akhtar, J. Chem. Soc., Chem. Commun. p. 643 (1974).
[43]
STEREOSPECIFIC PORPHOBILINOGENS
381
2H zH 3HTCOOMe > 3H-y COOMe >3H_yCOOH HOOC /
HOOC ,jI'-2H
'H~ - ~ O ~NH 2
Fxo. 4. Stereospecific introduction of hydrogen atoms at C-2 of ALA. In the first step, stereospecific reduction of the tritiated substrate in ZH20 using 2-enoate reductase gives methyl succinate which is then converted to (2S)-[2-3H,2-ZH]ALAchemically.
The stereospecifically labeled ALA thus formed may be trapped to give PBG stereospecifically labeled in the aminomethyl sidechain. 3.~5The preparative procedure is essentially similar to the coupled e n z y m e system described above, except that incubation conditions were chosen 3 to maximize the radiochemical incorporation of glycine rather than succinyl CoA, by reducing the glycine concentration from about 130 to 3 mM (Kin about 12 raM), and the reaction was performed in phosphate buffer rather than the Tris buffer. The complications arising from the nonenzymatic racemisation of the chiral center at C-5 of ALA catalyzed by pyridoxal phosphate (required as cofactor by the A L A synthetase) were avoided by using as low a concentration of pyridoxal phosphate as possible. Furthermore the system was loaded with ALA dehydratase to reduce the life of A L A in solution. Careful control of the thiol concentration was also felt to be important (for further details, see Ref. 16). It should be noted that this method has only ever been carried out on a small scale and is not suitable for deuterium labeling (Fig. 5). Reagents
[2RS-3Hz]Glycine (3/~mol, freeze-dried powder) Pyridoxal phosphate, 60/~M in distilled water T r i s - H C l buffer, 10 mM Potassium phosphate buffer, pH 6.8, l0 mM prepared in glycerol : water (2 : 8) which was also 2 m M in 2-mercaptoethanol Potassium phosphate buffer, pH 6.8, 10 m M which was also 20 mM in 2-mercaptoethanol Succinyl-CoA (3.75/~mol in 150 ~1 prepared as in Ref. 8) A L A synthetase and A L A dehydratase Dowex acetate, 2-X8 (400 mesh) 15C. Jones, P. M. Jordan, A. G. Chaudhry, and M. Akhtar, J. Chem. Soc., Chem. Commun. p. 96 (1979). ~6For further details, see C. Jones, Ph.D. Thesis, University of Southampton (1979).
382
HEME PORPHYRINS AND DERIVATIVES
[43]
COOH COOH
HR~ Hsi Fro. 5. PBG stereospeciflcal]y labeled at the aminomethyl group.
ALA synthetase and ALA dehydratase were prepared as described in Refs. 8 and 9. Succinyl-CoA was prepared from unlabeled succinic anhydride as described in Ref. 8. ALA synthetase 9 was dialyzed for 3 hr against 100 ml of the potassium phosphate buffer, pH 6.8 (10 mM) containing glycerol (20%) and 2-mercaptoethanol (2 mM) before use. ALA dehydratase8 was dissolved in a small volume of 10 mM potassium phosphate buffer containing 20 mM 2-mercaptoethanol and dialyzed against this buffer for 3 hr prior to use. The incubation mixture was prepared from [2-3H2]glycine (3 /xmol), pyridoxal phosphate (0.1 ml), Tris-HC1 buffer (0.1 ml), ALA synthetase (20 units), and ALA dehydratase (20 units) made up to 1 ml with distilled water. The final phosphate and thiol concentrations were estimated at 8 and 7 mM, respectively. The solution was warmed to 37° and reaction started by the addition of succinyl-CoA (1.25 ~mol in 50/xl; for preparation see above): further aliquots were added after 10 and 20 min. After 30 min reaction was stopped by applying it to a column of Dowex acetate (2 x 8,400 mesh; 0.5 x 6 cm) and the PBG isolated and further purified by TLC as above. The PBG from the ion exchange column was typically 98% radiochemically pure (impurities were ALA and glycine) and could often be used without further purification. Isolated radiochemical yield of the [2,11S-3H2]PBG was typically 30%. The enantiomeric excess of the product was about 84% as assayed using the protocol described in Ref. 3 (also see Ref. 17).
Variations [IIS-3HI]PBG. Dilute acid treatment of the PBG leads to exchange of the H-2 tritium in the doubly labeled PBG above. The labeled PBG (1 mg) 17 L. Schirch, this series, Vol. 17, Part B, p. 335; P. M. Jordan and M. Akhtar, Biochem. J. 116, 277 (1970).
[44]
HPLC
OF URO- AND COPROPORPHYRIN
ISOMERS
383
was dissolved in 0.15 M hydrochloric acid (100/A) and allowed to stand at room temperature for 1 hr, freeze dried, and the [11S-3H1]PBG purified by T L C on cellulose. Acknowledgments We thank SERC for their support of our work in the area of porphyrin biosynthesis.
[44] H i g h - P e r f o r m a n c e Uroporphyrin
Liquid Chromatography
and Coproporphyrin
of
Isomers
B y C. K. LIM and T. J. PETERS
Uroporphyrinogen III, the universal precursor of heme, chlorophylls, and vitamin Bt2, is derived from porphobilinogen (PBG) by the sequential action of porphobilinogen deaminase (hydroxymethylbilane synthase, EC 4.3.1.8) which catalyzes the formation of hydroxymethylbilane (HMB) from four molecules of PBG and uroporphyrinogen III synthase (EC 4.2.1.75) which converts HMB into uroporphyrinogen IlI. j A small amount uroporphyrinogen I is also formed by the spontaneous chemical ring-closure of HMB. The stepwise decarboxylation of the acetic acid groups of uroporphyrinogens I and III form the corresponding coproporphyrinogen isomers 2 and is catalyzed by uroporphyrinogen decarboxylase (EC 4.1.I.37). Porphyrins are formed by oxidation of the porphyrinogens. High-performance liquid chromatography (HPLC) has been shown to be the most effective technique for the separation of uro- and coproporphyrin isomers 3 (Fig. 1). The methyl esters of the isomers can be resolved on silica 4-6 and aminopropyl silica 7 or the porphyrin free acids can be
A. R. Battersby, C. J. R. Fookes, K. E. Gustafson-Potter, E. McDonald, and G. W. J. Matcham, J. Chem. Soc., Perkin Trans. l, p. 2427 (1982). 2 A. H. Jackson, H. A. Sancovich, A. M. Ferramola, N. Evans, D. E. Games, S. A. Matlin, G. H. Elder, and S. G. Smith, Philos. Trans. R. Sot'. London, Ser. B 273, 191 (1976). 3 C. K. Lim, J. M. Rideout, and D. J. Wright, J. Chromatogr. 282, 629 (1983). 4 H. Nordlov, P. M. Jordan, G. Burton, and A. I. Scot, J. Chromatogr. 190, 221 (1980). 5 A. H. Jackson, K. R. N. Rao, and S. G. Smith, Biochem. J. 203, 515 (1982). 6 A. H. Jackson, K. R. N. Rao, and S. G. Smith, Biochem. J. 207, 599 (1982). 7 I. C. Walker, M. T. Gilbert, and K. Stubbs, J. Chromatogr. 202, 491 (1980).
METHODS IN ENZYMOLOGY, VOL. 123
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