Phoratoxin, a Toxic Protein froin the Mistletoe Phoradendron tomentosum subsp. macrophyll urn (Loranthaceae)

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1 Em. J. Biochem. 32, (1973) Phoratoxin, a Toxic Protein froin the Mistletoe Phoradendron tomentosum subsp. macrophyll urn (Loranthaceae) Improvements in the Isolation Procedure and Further Studies on the Properties S. Tore MELLSTRAND and Gunnar SAMUELSSON Farmakognostiska Institutionen, Farmaceutiska Fakulteten, Stockholm (Received March 9/June 22, 1972) In a simplified procedure for isolation of phoratoxin, the dried plant material (stems and leaves) was extracted with 20//, aqueous acetic acid. The extract was treated with polyamide to remove colored impurities and subjected to gel filtration on Sephadex G-25. The high-molecular-weight fraction was chromatographed on SE-Sephadex, using a gradient elution technique, whereby pure phoratoxin was obtained. The material was homogeneous by standard criteria of protein purity. The molecular weight, determined in the ultracentrifuge by the sedimentation equilibrium method, is Phoratoxin has the following amino acid composition (residues/mole) : alanine 2, arginine 3, aspartic acid 4, 1/2-cystine 6, glycine 6, histidine 1, Isoleucine 3, leucine I, lysine 4, phenylalanine 1, proline 2, serine 5, threonine 5, tryptophan 1, tyrosine 1, valine I. The molecular weight, calculated from the amino acid composition, is The material contains no reducing sugars, amino sugars or sialic acids. The LD,,, determined by intraperitoneal injection in mice, is 0.57 _t 0.05 mg/kg bodyweight. Phoratoxin was isolated from Phoradendron tomentosum (DC) Engelm. subsp. macrophyllum (Cockerell) Wiens [ = Phoradendron serotinum (Raf.) M. C. Johnst.] by Samuelsson and Ekblad in 1967 [I]. Pharmacological experiments [2] showed phoratoxin to produce hypotension, bradycardia, negative inotropic effect on the heart muscle and vasoconstriction of vessels in skin and skeletal muscle. This paper describes the purification of phoratoxin by chromatography on SE-Sephadex, further properties of the protein, and a simplified isolation procedure. EXPERIMENTAL PROCEDURES Materials All chemicals used were of reagent grade unless otherwise stated. The plant material was of the same batch which had previously been used for isolation of phoratoxin [l]. Sephadex G-25, SE-Sephadex C-50 and C-25 (snlfoethyl Sephadex) were obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). Coomassie brilliant blue R 250, was a gift from Imperial Chemical Industries (ICI), England. Polyamide powder was prepared as previously described [I] from granules of Maranyl Nylon compound type A 100 obtained from Imperial Chemical Industries (ICI), England. Purification by Chromatography on SE-S ephadex C-25 of Phoratoxin Isolated by the Old Procedure 0.24 g phoratoxin (= fraction Seph I, CMC B peak I11 [I]) was dissolved in 2 ml 0.15 M sodium acetate buffer ph 5.0 and the solution applied to a column (7 x 130 mm) of SE-Sephadex, previously equilibrated with the same buffer. The column was eluted with a linear gradient of sodium acetate buffer from 0.35M ph 5.5 to 0.40 M ph 6.5 in a total volume of 21. The gradient was obtained by an arrangement described by Parr, as cited by Bock and Ling [3]. Fractions of 5 ml were collected and their protein content was determined from the absorbance at 280 nm. Phoratoxin was eluted between 725 and 1OOOml of effluent. After desalting on a column of SephadexG-25 and freeze drying, the yield of phoratoxin was 160 mg (66010). A Simplified Procedure jor Isolation of Phoratoxin The plant material was extracted with 201, acetic acid and the extract purified by treatment with polyamide as previously described [l]. Gel filtration was performed on Sephadex G-25 essentially as in the original procedure [I] but with the following modifications. The polyamide-treated extract was concentrated to 1OOOml instead of to

2 144 Isolation and Properties of Phoratoxin Eur. J. Aiochem. 250ml. 500ml of this solution was applied to the SephadexG-25 column. Aliquots of 20~1 of the fractions eluted before the appearance of C1- ions, were subjected to alkaline hydrolysis and determination of ninhydrin color [17]. One peak was obtained, followed by a steady increase in color yields towards C1--containing fractions. The first eluted peak was found to contain the total toxicity of the extract. Material from this peak was recovered by lyophilization and denoted as fraction IA. Yield 2.8 g from 100 g dry plant material. 2.0 g of fraction IA was dissolved in 20 ml0.025m phosphate buffer ph5.05 and the solution applied to a column (70 x 2 cm) of SE-Sephadex C-50 equilibrated with 0.05 M phosphate buffer ph 5.05 containing 0.020/, NaN, as a preservative. Elution was started with the same buffer and fractions of 15 ml were collected. When 1200 ml had passed the column, the eluant was changed to a phosphate buffer gradient, linearly changing from 0.05 M ph 5.05 to 0.3 M ph 6.0, in a total volume of 2000 ml [3]. The protein content of the eluted fractions was determined from the absorbance at 280nm. Fractions corresponding to protein-containing peaks were pooled, desalted on a column of Sephadex G-25, with 2O/, acetic acid as eluant and lyophilized. Phoratoxin was eluted between 2175 and 2325ml of effluent (Fig.1). Yield: 30 mg = 1.50/, of fraction IA. Oxidation of Phoratoxin Oxidation with performic acid was performed at 0 "C as described by Hirs [4]. High- Voltage Electrophoresis The method described by Samuelsson [5] was used with Sephadex 6-25 as supporting phase. Disc Electrophoresis The system of Reisfeld et al. [GI was used without sample and spacer gel. The sample was dissolved in a 50/, aqueous sucrose solution. 20-GO pg protein in a volume of pl was applied directly on the gel as a layer under the anode buffer. Electrophoresis was performed for 20 min at 50 V. The voltage was then increased to 300V for 406. Staining was performed with Coomassie brilliant blue R 250 according to Chrambach et al. [7]. The stained gels were stored in small test tubes filled with water and kept in the refrigerator. Quantitative Amino-Acid Analysis Proteins were hydrolyzed with constant boiling HC1 at 110 "C as described by Hirs et al. [S]. Following hydrolysis, the HC1 was removed in vacuo in a rotary evaporator and the amino acids determined with an automatic amino acid analyzer according to Spackman et al. [9], modified by Samuelsson [lo]. Determination of Tryptophan Tryptophan was determined in alkaline hydrolysates of phoratoxin, prepared according to Robe1 [ll]. Determination was performed on the amino acid analyzer, using a column (9 x 200 mm) filled with AminexA4 resin [lo], eluted with 0.35N sodium citrate buffer ph 5.35 at 50 "C. Tests for Sugars The presence of non-nitrogeneous sugar was tested by the Winder orcinol-sulphuric acid procedure [12]. For amino sugars the Rondle and Morgan method was used [13]. The method of Warren [14] was used for investigating the presence of sialic acids. Determination of LD,, The toxicity tests were performed on white mice as previously described [15]. Ultracentri fugation A Beckman-Spinco Model E analytical ultracentrifuge was used, equipped with schlieren optics and a photoelectric scanner accessory. Phoratoxin was dissolved in 0.1 M phosphate bueeri-ph 7.0. Sedimentation velocity experiments were performed at a speed of rev./min and a concentration of 7 mg phoratoxinlml in a synthetio boundary cell at 20 "C. Sedimentation equilibrium experiments were run according to Yphantis [16] at a speed of rev./min for 20 h at 20 "C. The concentration was 1.5 mg of phoratoxin/ml. Both scldieren and ultraviolet optics were used for registration. Edman Degradation Amino-terminal amino acids were chromatographically identified as phenylthiohydantoins following Edman degradation as described in [17]. RESULTS AND DISCUSSION Isolation of Phoratoxin The previous isolation procedure [l] for phoratoxin comprised the following steps: (a) extraction of the plant material with 201, acetic acid; (b) removal of colored substances by passage of the extract through a column of polyamide; (c) gel filtration, yielding a high-molecular-weight fraction denoted as fraction Seph I; (d) chromatography of fraction SephI on CM-cellulose, yielding a fraction of basic proteins containing all the toxicity of fraction Seph I,

3 Vol. 32, No. 1, 1973 S.T. MELLSTRAND and G. SAMUELSSON 145 Effiuent (mi) Fig. 1. Chromatography of 2.0 g fraction IA on a column of SE-Sephadex (7-50 (70 x 2 cm). (-) Absorbance at 280 nm of eluted fractions; (----) buffer gradient. Phoratoxin was eluted between 2175 and 2325 ml effluent and (e) chromatography of the basic proteins on phosphocellulose, whereby phoratoxin was obtained. The phoratoxin obtained by this procedure was homogeneous in rechromatography experiments and electrophoresis in a block of Sephadex. Lysine was the only N-terminal amino acid found. However, inhomogeneity of the preparation was suggested by a rather broad band in disc electrophoresis experiments and the appearance of three bands on electrophoresis of oxidized phoratoxinin a block of Sephadex [I]. This suspicion was confirmed when phoratoxin was subjected to chromatography on SE-Sephadex. One main peak and four smaller peaks were obtained. The yield of the material from the main peak was 66O/,. Based on this result improvements in the isolation procedure could be made. The improvements comprise steps (c, d, e) in the scheme above. In step (c) the load on the Sephadex G-25 column was decreased to half the amount of plant extract previously used, thereby ensuring better separation. It was also found that fraction Seph I could be divided into two fractions, only one of which, fraction IA, contained toxic proteins. Steps (d) and (e) were omitted and fraction IA was directly chromatographed on SE- Sephadex. The result is presented in Fig. 1. Homogeneity and Molecular Weight The following homogeneity tests were performed on phoratoxin isolated by the old procedure and purified by chromatography on SE-Sephadex. Phoratoxin gave only one peak in rechromatography on SE-Sephadex. Only one band was obtained in discelectrophoresis (Fig. 2). Following performic acid oxidation, phoratoxin gave only one band on highvoltage electrophoresis in a block of Sephadex G Eur. J. Riochrm.. Vol.32 Fig. 2. Polyucrylumide-gel electrophoresis of phorutoxia. The uample contained 20 pg protein. Stained with Coomassie brilliant blue R 250 In ultracentrifugation sedimentation velocity experiments (Fig. 3) phoratoxin behaved as a homogeneous substance with a sedimentation constant of ~ 2 0 =, 0.93 ~ S at 20 "C. The molecular weight was determined in a sedimentation equilibrium experiment, using ultraviolet optics at 280nm for determination of the weight-average molecular weight Mw, and schlieren optics for determination of the z-average molecular weight, M,. In both cases the molecular weight distribution throughout the cell formed a straight line (Fig.4) with M, = M, = 5000, using the value 0.71 for the partial specific volume. This value was calculated from the amino acid composition as given in Table I. Only one amino acid, lysine, was found to be amino-terminal in phoratoxin. Thus the purified phoratoxin is homogeneous by standard criteria of protein purity. Properties of Phoratoxilz The amino acid composition of purified phoratoxin is presented in Table 1. The minimum molecular weight calculated from this composition is 4854, including the terminal molecule of water and assuming that no aspartic acid is present as asparagine.

4 146 Isolation and Properties of Phoratoxin Eur. J. Biochem. Fig. 3. Sedimentation-velocity pattern of phoratoxin. Medium : 0.1 M phosphate buffer ph 7.0. Concentration of protein : 7 mg/ml. Speed: rev./min. Temperature: 20 "C. The photos were taken at 1, 25, 65 and 121 min after reaching full speed of the centrifuge Ac r2 (cm2) Big. 4. Molecular-weight determination of phoratoxin bzl the sedimentation-equilibrium method. (A) Determination of the z-average molecular weight by registration with schlieren optics. (B) Determination of the weight-average molecular weight by registration with ultraviolet optics. Medium: 0.1 M phosphate buffer. Concentration of protein: 1.5 mg/ml. Speed: rev./min for 20 h at 20 "C Amino acid Table 1. Amino-acid composition of phoratozin Found Amoiint. Nearest integer res idueslmolecule Lysine Histidine Arginine Aspartic acid Threonine a Serinea Proline Glycine Alanine l/,-cystineb Valine Isoleucine c Leuoine c Tyrosine Phenylalanine Tryptophan* * Corrected for destruction during hydrolysis. Determined as cysteic acid. 0 After 72 h hydrolysis. d After alkaline hydrolysis. This value is in good agreement with the value 5000 calculated from ultracentrifugation sedimentation equilibrium data as described above. No sugar, amino sugar or sialic acids were detected. The LD,, of phoratoxin, administered intraperitoneally to mice, was 0.57 & 0.05 mg/kg body weight. Identity of Phoratoxin Isolated by the Old Method Purified by Chromatography on SE-Sephadex and Phoratoxin Isolated by the Simplified Procedure Both preparations gave only one peak on rechromatography in the system used for isolation. The amino acid composition of the two preparations were exactly the same. A mixture (1:i) of the two preparations produced only one band in disc electrophoresis. The LD,, of the two preparations were the same. These results prove the identity of the material obtained by the two procedures.

5 V01.32, No.1, 1973 S.T. MELLSTRAND and G. SAMUELSSON 147 Comparison of Phoratoxin and the Viscotoxins Phoratoxin has previously been shown to have similar pharmacological effects as the viscotoxins isolated from the european mistletoe Viscum album L [2]. The chemical and physical properties described here and in the preceding paper [I], also show that phoratoxin is very similar to the viscotoxins [17,18]. Thus the molecular size and the total number of amino acid residues is the same in both phoratoxin and the viscotoxins. The number of disulfide bridges is probably the same, as phoratoxin also contains six half-cystine residues. Differences are indicated by the presence in phoratoxin of the amino acids histidine and tryptophan, which have not been found in the viscotoxins. This work was supported by grants No. B69-13X A and B70-13X B from the Swedish Medical Science Research Council. The ultracentrifugation experiments were performed by Ing. Hikan Pertoft at the Department of Medical Chemistry, University of Uppsala, through the courtesy of Prof. Torvard Lament. This valuable help is hereby gratefully acknowledged. We also wish to express our thanks to Mr Jarmo Tulonen for skilful technical assistance. REFERENCES 1. Samuelsson, G. & Ekblad, M. (1967) Acta Chem. Scand. 21, Rosell, S. & Samuelsson, G. (1966) Tozicon, 4, Bock, R. M. & Ling, N. S. (1954) Anal. Chem. 26, Him, C. H. W. (1956) J. Biol. Chem. 219, Samuelsson, G. (1982) Svensk Farm. Tidskr. 66, Reisfeld, R. A, Lewis, U. J. & Williams, D. E. (1962) Nuture (London) 195, Chrambach, A., Reisfeld, R. A., Wyckoff, M. & Zaccari, J. (1967) Anal. Biochem. 20, Hirs, C. H. W., Stein, W. H. & Moore, S. (1954) J. Biol. Chem. 211, Spackman, D. H., Stein, 157.H. & Moore, S. (1958) Anal. Chenz. 30, Samuelsson, G. (1967) Svensk Kem. Tidskr. 80, Robel, E. (1967) Anal. Riochem. 18, FranGois, C., Marshall, D. & Neuberger, A. (1962) Biochem. J. 83, Rondle, C. J. M. & Morgan, W. T. J. (1955) Biochenz. J. 61, Warren, L. (1959) J. Bid. Chem. 234, Samuelsson, G. (1958) Svensk Farm. Tidskr. 62, Yphantis, D. A. (1964) Biochemistry, 3, Samuelsson, G., Seger, L. & Olson, T. (1968) Acta Chem. Scand. 22, Samuelsson, G. & Pettersson, B. M. (1971) Eur. J. Biochem. 21, 86. S. T. Mellstrand and G. Samuelsson Farmakognostiska Institutionen Lindhagensgatan 128, S Stockholm, Sweden 10.