Uroporphyrinogen I Synthase from Human Erythrocytes: Separation, Purification, and Properties of Isoenzymes (Heme Synthesis/Porphobilinogen Deaminase/Porphyria) K

Proc. Nati. Acad. Sci. USA Vol. 76, No. 12, pp. 6172-6176, December 1979 Biochemistry Uroporphyrinogen I synthase from human erythrocytes: Separation, purification, and properties of isoenzymes (heme synthesis/porphobilinogen deaminase/porphyria) K. MIYAGI*, M. KANESHIMA*, J. KAWAKAMI*, F. NAKADAt, Z. J. PETRYKAt, AND C. J. WATSONt Departments of *Clinical Pathology and Biochemistry, College of Health Sciences, University of the Ryukyus, Naha, Okinawa 902, Japan; and *University of Minnesota Medical Research Unit at Northwestern Hospital, Minneapolis, Minnesota 55407 Contributed by Cecil James Watson, September 10, 1979 ABSTRACT Uroporphyrinogen I synthase [porphobilinogen other reagents of analytical grade were obtained from Wako ammonia-lyase (polymerizing), EC 4.3.1.8] from human eryth- Purechemical Ind. Ltd. and Nakarai Chem. Ltd. Phosphate rocytes was separated into two active protein peaks (A and B) the present experiments were made on DEAE-cellulose, by ammonium sulfate fractionation, on buffers used throughout Sephadex G-100, and' on DEAE-Sephadex A-50 with a NaCl of K2HPO4 and KH2PO4. gradient. The final purification was 613 and 743 times for A and Enzyme Source. Human whole blood withdrawn for blood B. respectively. The corresponding yields were 2.2 and 3.4%. transfusion was kept at 4-6'C for 3-7 days after being with- Fraction A was separated further into two (Al and A2) active drawn. To 200 ml of whole blood were added 0.327 g of citric protein bands'and fraction B into three (B1, B2, and B3) on ana- acid, 2.63 g of sodium citrate, 0.251 g of sodium dihydrophos- lytical polyacrylamide disc gel electrophoresis. Bands Al and A2 were identical with B1 and B2; B3 represented a third isoen- phate, and 2.32 g of glucose. Whole blood (400 ml) obtained zyme. Molecular weights (mean ± SEM), measured by gel fil- from two persons was used as a starting material. tration and sodium dodecyl sulfate/polyacrylamide gel elec- Enzyme Assay. UPGI-S activity was assayed by measuring trophoresis, were 38,000 ± 1000 for B1 and 40,000 ± 1000 for B2 the formation of uroporphyrinogen as follows. The incubation and B3. Isoelectric focusing on 4% polyacrylarmide gel separated mixture contained 50 mM Tris-HCl at pH 8.2, 600 mM por- both fractions A and B into three active protein bands. Maximal phobilinogen, and the enzyme solution in a total volume of 1.5 activity of the enzyme was found in gel cuts (5-mm) at pH 5.6 ml. Incubation was at 370C for 3 hr. After the incubation, for both fractions A and B. protein was precipitated with 1.5 ml of 25% (wt/vol) trichlo- Two enzymes are involved in the cyclic polymerization of four roacetic acid. After 15 min in the dark, 0.1 ml of an ethanolic molecules of porphobilinogen into uroporphyrinogen III, solution of 0.2% I2 and 0.4% KI was added. The residual iodine uroporphyrinogen I synthase [UPGI-S; porphobilinogen am- was reduced by the addition of 0.1 ml of 0.4% K2S204, and the monia-lyase (polymerizing), EC 4.3.1.8] (1) and uroporphyri- formed porphyrin was measured fluorometrically; as a stan- nogen III cosynthase (2). UPGI-S acting alone on porphobili- dard, 10 jig of coproporphyrin in 12.5% trichloroacetic acid nogen forms uroporphyrinogen I. Uroporphyrinogen III co- solution was used. synthase by itself has no catalytic function on either porpho- An enzyme unit is defined as the amount of enzyme that bilinogen or uroporphyrinogen I but, in conjunction with catalyzes the formation of 1 ,ug of uroporphyrinogen per hour UPGI-S acting on porphobilinogen, it forms uroporphyrinogen under standard conditions. The specific enzyme activity is III, the natural precursor of heme and chlorophyll. expressed as number of enzyme units per mg of protein. UPGI-S has been isolated and purified from bacteria, higher Determination of Protein Concentration. Protein con- plants, and avian and mammalian erythrocytes (3-10). Al- centration was measured by the method of Lowry et al. (12). though the enzyme has been isolated from many different Protein concentration in samples containing 2-mercaptoethanol sources, the properties of purified UPGI-S are similar. The was determined after dialysis against distilled water. enzyme from mammalian tissue can be replaced in vitro by the Polyacrylamide Disc Gel Electrophoresis. The preparation enzyme from higher plants (6). This enzyme from many dif- of the gel and electrophoresis was according to the method of ferent sources also has similar molecular weights (3-5, 7, 11). Davis (13). The following modifications were introduced. The In all of the reports, the final purified enzyme preparation has sample solution was mixed with an equal volume of 40% been shown to be a single protein. However, little work has been (wt/vol) sucrose. The separation gel contained 5 mM phosphate done on the enzyme from human erythrocytes, and the prop- at pH 7.4, 0.1 mM 2-mercaptoethanol, and 0.3% glycerol. A erties of human UPGI-S have not yet been well defined. constant current of 2 mA per gel was applied until the protein The present paper describes the purification of UPGI-S from went into the separation gel; then the current was increased to human erythrocytes and its separation into three active pro- 3 mA per gel and the gel was maintained at 4VC for 2.5 hr. teins. The developed gel was cut into halves lengthwise. One half was stained for protein identification with 1% Amidoblack 1OB MATERIALS AND METHODS in 7% acetic acid for 30 min. Destaining was performed elec- Reagents. Porphobilinogen was purchased from Sigma. trophoretically to shorten the procedure. The other half was DEAE-cellulose (DE-23) was from Whatman Ltd. Sephadex kept in a small amount of 0.05 M phosphate buffer at pH 7.4 G-100, DEAE-Sephadex A-50, and marker proteins for the at room temperature. Comparison of the halves indicated the from Phar- desired protein band to be cut for isolation. molecular weight determinations were obtained Proteins were eluted electrophoretically by the method of macia. Reagents for polyacrylamide disc gel electrophoresis and Braatz and McIntire (14). The buffer used for protein elution was 0.134 M mM The publication costs of this article were defrayed in part by page phosphate, pH 7.4/1 2-mercaptoethanol. charge payment. This article must therefore be hereby marked "ad- Electrophoresis was carried out with constant current (2 mA vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation UPGI-S, uroporphyrinogen I synthase. 6172 Downloaded by guest on September 24, 2021 Biochemistry: Miyagi et al. Proc. Natl. Acad. Sci. USA 76 (1979) 6173 per tube) at 40C for 4 hr. The sliced gels and the protein eluates equilibration of the Sephadex and the elution were carried out were passed through a nylon filter, the column was washed with the same buffer. The eluates containing enzyme activity several times with small amounts of eluting buffer, anfdthe were combined. eluates were combined. Heat Treatment. The enzyme preparation from the above Isoelectric Focusing on 4% Polyacrylamide Disc Gel. The step was heated at 60°C for 15 min, cooled in an ice bath, and separation gel solution was prepared by mixing 3.2 ml of solu- centrifuged at 10,000 X g for 10 min. The precipitate was dis- tion A, 3.0 ml of solution B, 1.0 ml of 0.5% ammonium persul- carded. fate solution, 1.2 ml of 40% Ampholine (pH 3.5-10), and 15.6 DEAE-Sephadex A-50. The supernate after heat treatment ml of 5 mM phosphate, pH 7.4/0.1 mM 2-mercaptoethanol/ was concentrated in a collodion bag to approximately 5 ml and 0.3% glycerol. Solutions: A, 30 g of acrylamide and 1.5 g of then applied to a column (1.5 X 60 cm) of DEAE-Sephadex methylenebisacrylamide dissolved in water (total volume, 100 A-50 preequilibrated with 0.05 M phosphate, pH 7.4/0.01 M ml); B, 4 mg of riboflavin and 0.45 ml of N,N,N',N'-tetra- 2-mercaptoethanol. Elution was carried out with the same methylenediamine dissolved in water (total volume, 100 ml). buffer containing 0.2 M NaCl. The sample solution consisted of 1 vol (300 ,ug) of enzyme so- Rechromatography on DEAE-Sephadex A-50 with Linear lution and 1 vol of the mixture of 20% glycerol and 4% Am- NaCI Gradient. The enzyme preparation from the above step pholine (pH 3.5-10). The sample-protecting solution contained was concentrated in a collodion bag to approximately 5 ml. 1.5 ml of glycerol and 40% Ampholine per 100 ml. The glass Without dialysis, the enzyme solution was applied to a column tube (10 X 0.5 cm inside diameter) was filled with 7 ml of of DEAE-Sephadex A-50 (1.5 X 60 cm) preequilibrated with separation gel solution. After 30-40 min, no more than 50 ul 0.05 M phosphate, pH 7.4/0.01 M 2-mercaptoethanol. The of sample solution (desalted through Sephadex G-25) was ap- concentration of NaCl was increased linearly from 0 to 0.2 plied on the top of the gel, and then protection solution was M. layered on. The electrophoresis was carried out at 200 V at 4VC Determination of Molecular Weight. The molecular for 3 hr. weights of enzyme preparations were determined by two One of the simultaneously run gels was stained for proteins methods. (i) The gel filtration method of Andrews (16). A and the other one was cut into 5-mm-long sections. Each gel Sephadex G-100 column (1.5 X 70 cm) was preequilibrated segment was put in 5 ml of distilled water in a capped test tube with 0.05 M phosphate, pH 7.4/1 mM 2-mercaptoethanol.

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