- Email: [email protected]
[23] Preparation of Yersinia pestis plague murine toxin
162
PREPARATION OF TOXINS
[23]
1 : 1000 in PBS-T, is added for 1 hr at room temperature. Wells are again washed and the reaction developed by addition of enzyme substrate [0.2 ml ofp-nitrophenyl phosphate, 1 mg/ml in diethanolamine buffer, pH 9.8 (Sigma Chemical Co.)] for 1 hr at room temperature. The absorbance in each well is measured at 405 nm with an automated ELISA reader. Net absorbance is determined by subtracting the absorbance in wells treated with PBS-T in place of test sample from the absorbance in well with toxincontaining test samples. A standard curve is constructed using pure toxin, and unknown samples can be assayed by plotting results from serial dilutions of the unknown on the standard curve. Using the antitoxin antibodies we have described, 17this assay gives reproducible results with as little as 10 pg toxin/well.
Acknowledgment The work in our laboratory was funded by Grants AI-16242, AI-20325,and AM-39428, from the National Institutes of Health, Bethesda, Maryland, Grant 82008 from the Programme for Control of Diarrhoeal Diseases, World Health Organization, Geneva, Switzerland, and a grant in geographicmedicinefrom the RockefellerFoundation, New York, NY.
[23] P r e p a r a t i o n o f Yersinia pestis Plague Murine Toxin
By THOMAS C. MONTIE
Introduction Plague murine toxin appears to be an envelope protein component of the bacterium Yersinia pestis, formerly Pasteurella pestis. As isolated, the toxin may contain one or usually two related protein species of different molecular weights (120,000 and 240,000). At the end of the growth cycle toxin is released into the medium following autolysis of the cells. The method described below utilizes this observation to facilitate toxin isolation, although toxin also can be released following sonication of the bacterial envelope.1 The two proteins isolated are toxic for mice and rats 1 T. C . M o n t i e a n d S. J. A j l , J. Gen. Microbiol. 34, 249 (1964).
METHODS IN ENZYMOLOGY, VOL. 165
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
[23]
Yersinia pestis PLAGUETOXIN
163
at low levels (0.1-1.0/zg/20 g mouse). Mouse lethality then must be an important assay for ultimately determining the biological activity of the protein product. Immunodiffusion and gel electrophoresis assays also serve to facilitate routine assays of the toxin. Although both toxin A and B appeared routinely in the Y. pestis strain Tijwidej (TJW) preparations, toxin A seemed to be the most biologically active of the two proteins. Also, a survey of different avirulent strains indicated it to be the most predominant if not the only toxin present in some Y. pestis extracts. It is difficult to explain the toxicity in terms of any unusual feature of the primary sequences of the protein. However, the sulfhydryl and tryptophan residues are important for toxicity. 2 Evidence to date indicates that the toxin acts as a beta blocking agent (see review by Montie3). Isolation and Preparation
Growth of Cells A number of different strains of avirulent Y. pestis have been used to prepare toxin. We have generally used strain TJW maintained on agar (1.6%) slants containing casein hydrolysate or casamino acids plus mineral salts as modified from Englesberg and Levy. 4 This same medium (CHMG) is used to grow up to 7 liters of batch culture to be used for isolation. The CHMG medium contains the following, in grams per liter: casein hydrolysate or casamino acids (30.0), NH4CI (1.0), MgSO4"7H20 (0.5), CaCI: finely ground (0.01), and FeCI3 • 6H20 (0.0025) or (1.0 ml from a stock solution containing 0.25 g/100 ml H20). Stock solutions of potassium phosphate and glucose are autoclaved and added separately; final concentrations are potassium phosphate (0.05 M) and D-glucose (2%). A 24-hr-old CHMG slant culture is washed into 250 ml of the 7 liters to be used as starter culture. The latter culture is grown at 27-28 ° for 24 hr on a shaker. The starter is inoculated into a 9-liter carboy containing the remaining 7 liters of medium. This 7-liter culture is aerated and stirred by means of a finely performed, bubbling stone apparatus connected to forced air for 7 days at room temperature (maximum toxin release occurs after approximately 5 to 7 days). Antifoam may be added if necessary. Cells and cell debris are then removed by centrifugation for 30 min at 9000 rpm (13,000 g) at 4 ° and the supernatant is saved for processing. 2 T. C. Montie and D. B. Montie, Biochemistry 12, 4958 (1973). 3 T. C. Montie, Pharmacol. Ther. 12, 491 (1981). 4 E. Englesberg and S. B. L e v y , J. Bacteriol. 68, 57 (1954).
164
PREPARATION OF TOXINS
[23]
Ammonium Sulfate Fractionation--Preparation of Crude Toxin 1. The supernatant is brought to 100% saturation (70 g/100 ml) with ammonium sulfate and stirred slowly in the cold overnight. The precipitated toxin is centrifuged or removed with a beaker from the surface of the culture if floating. The precipitate is dissolved in a small amount of distilled water and dialyzed against water with a number of changes until no BaSO4, precipitate is detected with Ba(OH)2 [two drops saturated Ba(OH)2 solution/ml crude toxin solution]. 2. Ammonium sulfate is again added slowly with stirring at 4° until 35% saturation is reached (24.5 g/100 ml). The precipitate is removed by centrifugation and discarded. 3. The supernatant containing the toxin is brought slowly to approximately 70% saturation by addition of ammonium sulfate at 4°. The solution can be stored overnight at 4° to obtain the maximum amount of precipitated toxin. The toxin precipitate is removed by centrifugation (4°), redissolved in a small amount of water, and dialyzed exhaustively against water and lyophilized. The crude toxin preparation is best stored desiccated at - 7 0 ° to avoid denaturation.
Purification and Resolution of Toxins A and B by Molecular Seiving The crude toxin, containing two toxic proteins as major components, can be further purified by molecular seiving procedures using BioGel or Sephadex columns. 5 Typically, 4.0 mg of crude toxin is dissolved in 2 ml 0.01 M potassium phosphate buffer (pH 8.0) and centrifuged to remove any undissolved or denatured toxin materials. The toxin sample is passed through a Sephadex G-100 or G-200 column (2.5 × 40 cm) which has been previously equilibrated with the pH 8.0 phosphate buffer. Elution of toxin is detected at 280 nm. Toxin A (240 kDa) appears at the void volume (eluate volume of approximately 58 ml in Sephadex G-100) and continues completely resolved from toxin B (120 kDa) for approximately 6 to 10 ml of eluate. Both toxins elute next in the trailing position of the first peak, and toxin B emerges as a distinct shoulder trailing the first major ultraviolet-positive peak. The ultraviolet pattern does not clearly distinguish the resolution of the two toxins so that assay by disc or slab gel electrophoresis (PAGE) or by immunodiffusion using immune rabbit serum 5 is required. Toxin obtained exhibited activity in 16- to 18-g female mice Swiss albino mice of an approximate LDs0 of 1 ~g following intraperitoneal injection. Fractions are pooled and dialyzed with a final dialysis step against 0.001 M phosphate buffer before lyophilization. The latter step 5 T. C. Montie, D. B. Montie, and S. J. Ajl, Biochim. Biophys. Acta 130, 406 (1966).
[23]
Yersinia p e s t i s PLAGUE TOXIN
165
aids in preventing denaturation. Storage of the dried material should be at - 7 0 ° desiccated. Isolation by Polyacrylamide Gel Electrophoresis Elution of Toxin from Acrylamide Gels 6 Some of the earliest isolation techniques involved elution of toxin protein from disc gels. This technique could be readily applied to slab gel preparation to obtain greater amounts in continuous or discontinuous systems. 7,8 The disadvantage of the disc gel technique is that only microgram amounts of toxin can be obtained. In this technique crude toxin between 1 and 2 mg is loaded onto 0.8 x 5 cm acrylamide disc gelcolumns placed in cut sections of 10-ml pipets following the general method of Davis. 9 The run is carried out in an alkaline Tris-HCl buffer (pH 8.9). With the toxins both sample and spacer gels can be eliminated and 20% sucrose or Sephadex are acceptable substitutes for loading the sample. Toxin is premixed with these components and layered onto the small-pore running gel. Parallel disc gel or slab gel lanes are used to locate by the location of toxin following 0.5 hr of rapid staining with Amido black or Coomassie blue. For elution unstained parallel discs or slab sections are cut into 2- to 3-mm sections (up to 6 mm for long slab gels) and eluted with either 0.05 sodium maleate buffer (pH 6.6) or 0.01 M phosphate buffer (pH 7.6) at 4 °. The gel sections are agitated during the elution period lasting 24 to 48 hr. In some cases gels may be eluted directly by encasing the slice in dialysis tubing. An approach somewhat more time consuming, but affording a higher yield, involves homogenization of the gel slice in buffer using a motor-driven Teflon pestle. The homogenates are then centrifuged and the supernatants dialyzed against distilled water. Although the slightly acid buffer resulted in elution of lower amounts of toxin compared to phosphate elution at this pH it seemed to deter elution of nonspecific UV-absorbing material which was more evident in alkaline buffers. Protein eluted was toxic and could be detected by immunodiffusion. For quick assay to detect toxin, entire uneluted gel sections placed in antigen wells were adequate sources of antigen for production of immunoprecipitin bands. 6 T. C. Montie, D. B. Montie, and S. J. Ajl, J. Exp. Med. 120, 1201 (1964). 7 "Hoefer Scientific Instruments Catalogue," p. 96. Hoefer Scientific Instruments, San Francisco, California, 1983. 8 A. Chrambach and D. Rodbard, Science 172, 440 (1971). 9 B. J. Davis, Ann. N.Y. Acad. Sci. 121, 404 (1964).
166
PREPARATION OF TOXINS
[23]
Preparative Gel Electrophoresis 1o A large preparative gel electrophoresis [Poly-Prep 100, Biachler Instruments Div., Nuclear-Chicago Corporation (Searle and Co.), Fort Lee, N J]. The advantage of this technique is that milligram amounts of highly purified toxin can be obtained directly from the crude ammonium sulfate prepared toxin. For example, from 250 mg of crude toxin (two runs), 11.3 mg of purified toxin A was obtained. No evidence for lipopolysaccharide contamination was found in biological studies. 11 In some preparations a toxic dose was at the level of 0.1 tzg Lowry protein. Toxin (80 to 150 mg) sample is dissolved in up to 15 ml of sample buffer (2.89 tzg Tris and 12.8 ml 1 M H3PO4 brought to 100 ml) and layered onto a preformed concentrating gel using riboflavin-polymerized acrylamide (see components and solutions, Table I) or by directly layering a toxin solution of Tris-glycine or Tris-phosphate sample buffer with 10% sucrose onto a 6-cm resolving gel. One drop of concentrated bromphenol blue is added as a marker dye. The column is continuously cooled at 6-8 °. Run time is approximately 36 hr in a Tris-glycine pH 8.9, 7.5% acrylamide running gel system with an elution flow rate of 20 ml/hr. Toxin bands are identified on analytical gels using 0.02 ml of a 2- to 4-ml collected fraction. A constant amperage is required, set at 50 mA. The voltage will rise slowly during the run from 250 to 400-450 V. The individual toxins are resolved into two peaks of approximately 20 consecutive 2-ml fractions, for example, with toxin B in fractions 58 to 70, and toxin A in fractions 135 to 155 (see analytical gel profile in Ref. 10). The toxin samples are pooled, dialyzed against 0.001 M KHzPO4, pH 7.0, buffer, and lyophilized. Storage of lyophilized material is at - 7 0 °, desiccated. Preparation of Toxin Subunit Components
Isolation of Biologically Active Subunits Using Sodium Dodecyl Sulfate (SDS)12,13 Although large-molecular-weight murine toxin is partially denatured by treatment with 0.5-1% SDS, it apparently retains partial activity even though dissociation occurs to a subunit of approximate MW 24,000. These subunits may be obtained by dissolving toxin protein in 1.0% SDS and incubating at 37 ° for 3 hr. Excess SDS is removed by dialysis against 0.1% ~0T. C. Montie and D. B. Montie, J. Bacteriol. 100, 535 (1969). 11 S. D. Brown and T. C. Montie, Infect. Imrnun. 18, 85 (1977). 12 T. C. Montie, D. B. Montie, S. A. Leon, C. A. Kennedy, and S. J, Ajl, Biochern. Biophys. Res. Commun. 33, 423 (1968). 13 T. C. Montie and D. B. Montie, Biochemistry 10, 2094 (1971).
Yersinia pestis PLAGUE TOXIN
[23]
167
TABLE I REQUIRED SOLUTIONS FOR BUCHLER POLY-PREP ACRYLAM1DE GEL ELECTROPHORESlS Buffers
Chemicals
Amount
Concentration (-fold)
Upper (per 2 liters) Lower (per 2 liters) Membrane (per liter) Sample (per 100 ml) Elution
Tris Glycine Tris 1 N HC1 Tris 1 N HCI 1 M H3PO4 Tris Lower buffer H20
12.64 g 7.88 g 72.6 ml 300 ml 48.4 g 200 ml 12.8 ml 2.85 g 1 part 2 parts
--3× 4× --1×
Stock solutions
Gel
Volume desired
Resolving
100 ml
Volume ratio 25 ml 25 ml
50 ml Concentrating ~
50 ml
30ml 15 ml
Additional solutions
15 ml 100 ml H20 100 ml H20
Component Acrylamide Bisacrylamide 1 N HC1 Tris TEMED Ammonium persulfate Acrylamide Bisacrylamide 1 M H3PO4 Tris TEMED Riboflavin Sucrose Bromphenol blue
Concentration /t00 ml 30.0 2.0 24.0 18.15 0.2- 0.4 0.15
g g ml g ml g
5.0 0.3- 1.25 12.8 2.85 0.1 2.0 40 5.0
g g ml g ml mg g mg
" This gel is hardened using a bank of fluorescent lights with riboflavin as the catalyst.
SDS for 17 to 24 hr. Under these conditions at least 60% of the toxic activity is retained." To avoid dialysis, passage through a Sephadex G-75 or G-100 column will remove excess SDS. The subunit form is retained and identified as the major retarded peak. In more dilute incubation solutions a polypeptide of 10,000-12,000 Da is obtained; however, the toxic activity of this unit may be eliminated. Specific conditions for subunit isolation and/or molecular weight determination include passing 1 to 2 mg/ml toxin in SDS-phosphate through a Sephadex G-100 column (1.5 × 90 cm) equilibrated with 0.1 M sodium phosphate buffer (pH 7.1).
168
PREPARATION OF TOXINS
[23]
Isolation of Inactivated Subunits with Organic Acids: Citric Acid and Acetic Acid Plus EDTA lz
Under these conditions lethal activity is lost, although attempts to reverse the acid denaturation have not been explored fully. In this method toxin (1-4 mg) is rapidly dissolved in 0.5 to 2.0 ml of citric acid (0.1 M, pH 2.2) and incubated at 37° for 1 hr or 26 ° for 24 hr (partial depolymerization). Complete depolymerization to a 12,000-Da fragment occurs after exposure at 37° for 24 hr. Alternatively, 0.1 M acetic acid with 10-3 M EDTA can be substituted for citric acid with approximately the same result. Toxin subunits are isolated by passing toxin dissolved in acid through a Sephadex G-50 column equilibrated with 0.001 M potassium phosphate (pH 7.0) and 0.1 M KC1. The high salt is added if column adsorption is a problem, but usually it can be omitted. Excess acid may be removed by passing the pooled two or three peak fractions through a second G-50 column. Acetic acid is the acid of choice for peptide determinations since radioactive citrate is bound to toxin even after tryptic digestion and separation of peptides by high-voltage electrophoresis. Isolation of Inactive Polypeptides by Gel Electrophoresis in a Phenol-Acetic Acid-Urea GeP °
Lyophilized native toxin fractions from the Btichler Poly-Prep gel electrophoresis are further separated into polypeptides by gel electrophoresis in a denaturing environment. Microgram amounts of toxin A or B (5 to 25 tzg) are dissolved in 0.1 to 0.2 ml phenol-acetic acid-water (2 : 1 : 0.5, v/v/v) and subjected to electrophoresis for 2 hr at room temperature through 7.5% (w/v) acrylamide gels containing 35% (v/v) acetic acid and 5 M urea. Protein bands are stained in the usual way with Amido black or Coomassie blue. Toxin A gives two bands: the least electropositive is designated No. 1 and the more electropositive band is designated No. 2. Toxin B exhibits two bands: a band corresponding to the No. 2 band of toxin A, and an additional No. 3 band of greater electrophoretic mobility than the No. 2 band. The No. 2 band (common to both toxins) always appears as the heaviest, in greatest proportion, or most densely stained band of the three. This method has been used by Rotten and Razin ~4 to identify mycoplasma membrane proteins and by Takayama et al.15 to distinguish mitochondrial membrane proteins.
~4 S. Rottem and S. Razin, J. Bacteriol. 94, 359 (1967). 15 K. Takayama, D. H. MacLennon, A. Tzagoloft, and C. D. Stoner, Arch. Biochem. Biophys. 114, 223 (1966).
PREPARATION OF TOXINS
[23]
1 : 1000 in PBS-T, is added for 1 hr at room temperature. Wells are again washed and the reaction developed by addition of enzyme substrate [0.2 ml ofp-nitrophenyl phosphate, 1 mg/ml in diethanolamine buffer, pH 9.8 (Sigma Chemical Co.)] for 1 hr at room temperature. The absorbance in each well is measured at 405 nm with an automated ELISA reader. Net absorbance is determined by subtracting the absorbance in wells treated with PBS-T in place of test sample from the absorbance in well with toxincontaining test samples. A standard curve is constructed using pure toxin, and unknown samples can be assayed by plotting results from serial dilutions of the unknown on the standard curve. Using the antitoxin antibodies we have described, 17this assay gives reproducible results with as little as 10 pg toxin/well.
Acknowledgment The work in our laboratory was funded by Grants AI-16242, AI-20325,and AM-39428, from the National Institutes of Health, Bethesda, Maryland, Grant 82008 from the Programme for Control of Diarrhoeal Diseases, World Health Organization, Geneva, Switzerland, and a grant in geographicmedicinefrom the RockefellerFoundation, New York, NY.
[23] P r e p a r a t i o n o f Yersinia pestis Plague Murine Toxin
By THOMAS C. MONTIE
Introduction Plague murine toxin appears to be an envelope protein component of the bacterium Yersinia pestis, formerly Pasteurella pestis. As isolated, the toxin may contain one or usually two related protein species of different molecular weights (120,000 and 240,000). At the end of the growth cycle toxin is released into the medium following autolysis of the cells. The method described below utilizes this observation to facilitate toxin isolation, although toxin also can be released following sonication of the bacterial envelope.1 The two proteins isolated are toxic for mice and rats 1 T. C . M o n t i e a n d S. J. A j l , J. Gen. Microbiol. 34, 249 (1964).
METHODS IN ENZYMOLOGY, VOL. 165
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
[23]
Yersinia pestis PLAGUETOXIN
163
at low levels (0.1-1.0/zg/20 g mouse). Mouse lethality then must be an important assay for ultimately determining the biological activity of the protein product. Immunodiffusion and gel electrophoresis assays also serve to facilitate routine assays of the toxin. Although both toxin A and B appeared routinely in the Y. pestis strain Tijwidej (TJW) preparations, toxin A seemed to be the most biologically active of the two proteins. Also, a survey of different avirulent strains indicated it to be the most predominant if not the only toxin present in some Y. pestis extracts. It is difficult to explain the toxicity in terms of any unusual feature of the primary sequences of the protein. However, the sulfhydryl and tryptophan residues are important for toxicity. 2 Evidence to date indicates that the toxin acts as a beta blocking agent (see review by Montie3). Isolation and Preparation
Growth of Cells A number of different strains of avirulent Y. pestis have been used to prepare toxin. We have generally used strain TJW maintained on agar (1.6%) slants containing casein hydrolysate or casamino acids plus mineral salts as modified from Englesberg and Levy. 4 This same medium (CHMG) is used to grow up to 7 liters of batch culture to be used for isolation. The CHMG medium contains the following, in grams per liter: casein hydrolysate or casamino acids (30.0), NH4CI (1.0), MgSO4"7H20 (0.5), CaCI: finely ground (0.01), and FeCI3 • 6H20 (0.0025) or (1.0 ml from a stock solution containing 0.25 g/100 ml H20). Stock solutions of potassium phosphate and glucose are autoclaved and added separately; final concentrations are potassium phosphate (0.05 M) and D-glucose (2%). A 24-hr-old CHMG slant culture is washed into 250 ml of the 7 liters to be used as starter culture. The latter culture is grown at 27-28 ° for 24 hr on a shaker. The starter is inoculated into a 9-liter carboy containing the remaining 7 liters of medium. This 7-liter culture is aerated and stirred by means of a finely performed, bubbling stone apparatus connected to forced air for 7 days at room temperature (maximum toxin release occurs after approximately 5 to 7 days). Antifoam may be added if necessary. Cells and cell debris are then removed by centrifugation for 30 min at 9000 rpm (13,000 g) at 4 ° and the supernatant is saved for processing. 2 T. C. Montie and D. B. Montie, Biochemistry 12, 4958 (1973). 3 T. C. Montie, Pharmacol. Ther. 12, 491 (1981). 4 E. Englesberg and S. B. L e v y , J. Bacteriol. 68, 57 (1954).
164
PREPARATION OF TOXINS
[23]
Ammonium Sulfate Fractionation--Preparation of Crude Toxin 1. The supernatant is brought to 100% saturation (70 g/100 ml) with ammonium sulfate and stirred slowly in the cold overnight. The precipitated toxin is centrifuged or removed with a beaker from the surface of the culture if floating. The precipitate is dissolved in a small amount of distilled water and dialyzed against water with a number of changes until no BaSO4, precipitate is detected with Ba(OH)2 [two drops saturated Ba(OH)2 solution/ml crude toxin solution]. 2. Ammonium sulfate is again added slowly with stirring at 4° until 35% saturation is reached (24.5 g/100 ml). The precipitate is removed by centrifugation and discarded. 3. The supernatant containing the toxin is brought slowly to approximately 70% saturation by addition of ammonium sulfate at 4°. The solution can be stored overnight at 4° to obtain the maximum amount of precipitated toxin. The toxin precipitate is removed by centrifugation (4°), redissolved in a small amount of water, and dialyzed exhaustively against water and lyophilized. The crude toxin preparation is best stored desiccated at - 7 0 ° to avoid denaturation.
Purification and Resolution of Toxins A and B by Molecular Seiving The crude toxin, containing two toxic proteins as major components, can be further purified by molecular seiving procedures using BioGel or Sephadex columns. 5 Typically, 4.0 mg of crude toxin is dissolved in 2 ml 0.01 M potassium phosphate buffer (pH 8.0) and centrifuged to remove any undissolved or denatured toxin materials. The toxin sample is passed through a Sephadex G-100 or G-200 column (2.5 × 40 cm) which has been previously equilibrated with the pH 8.0 phosphate buffer. Elution of toxin is detected at 280 nm. Toxin A (240 kDa) appears at the void volume (eluate volume of approximately 58 ml in Sephadex G-100) and continues completely resolved from toxin B (120 kDa) for approximately 6 to 10 ml of eluate. Both toxins elute next in the trailing position of the first peak, and toxin B emerges as a distinct shoulder trailing the first major ultraviolet-positive peak. The ultraviolet pattern does not clearly distinguish the resolution of the two toxins so that assay by disc or slab gel electrophoresis (PAGE) or by immunodiffusion using immune rabbit serum 5 is required. Toxin obtained exhibited activity in 16- to 18-g female mice Swiss albino mice of an approximate LDs0 of 1 ~g following intraperitoneal injection. Fractions are pooled and dialyzed with a final dialysis step against 0.001 M phosphate buffer before lyophilization. The latter step 5 T. C. Montie, D. B. Montie, and S. J. Ajl, Biochim. Biophys. Acta 130, 406 (1966).
[23]
Yersinia p e s t i s PLAGUE TOXIN
165
aids in preventing denaturation. Storage of the dried material should be at - 7 0 ° desiccated. Isolation by Polyacrylamide Gel Electrophoresis Elution of Toxin from Acrylamide Gels 6 Some of the earliest isolation techniques involved elution of toxin protein from disc gels. This technique could be readily applied to slab gel preparation to obtain greater amounts in continuous or discontinuous systems. 7,8 The disadvantage of the disc gel technique is that only microgram amounts of toxin can be obtained. In this technique crude toxin between 1 and 2 mg is loaded onto 0.8 x 5 cm acrylamide disc gelcolumns placed in cut sections of 10-ml pipets following the general method of Davis. 9 The run is carried out in an alkaline Tris-HCl buffer (pH 8.9). With the toxins both sample and spacer gels can be eliminated and 20% sucrose or Sephadex are acceptable substitutes for loading the sample. Toxin is premixed with these components and layered onto the small-pore running gel. Parallel disc gel or slab gel lanes are used to locate by the location of toxin following 0.5 hr of rapid staining with Amido black or Coomassie blue. For elution unstained parallel discs or slab sections are cut into 2- to 3-mm sections (up to 6 mm for long slab gels) and eluted with either 0.05 sodium maleate buffer (pH 6.6) or 0.01 M phosphate buffer (pH 7.6) at 4 °. The gel sections are agitated during the elution period lasting 24 to 48 hr. In some cases gels may be eluted directly by encasing the slice in dialysis tubing. An approach somewhat more time consuming, but affording a higher yield, involves homogenization of the gel slice in buffer using a motor-driven Teflon pestle. The homogenates are then centrifuged and the supernatants dialyzed against distilled water. Although the slightly acid buffer resulted in elution of lower amounts of toxin compared to phosphate elution at this pH it seemed to deter elution of nonspecific UV-absorbing material which was more evident in alkaline buffers. Protein eluted was toxic and could be detected by immunodiffusion. For quick assay to detect toxin, entire uneluted gel sections placed in antigen wells were adequate sources of antigen for production of immunoprecipitin bands. 6 T. C. Montie, D. B. Montie, and S. J. Ajl, J. Exp. Med. 120, 1201 (1964). 7 "Hoefer Scientific Instruments Catalogue," p. 96. Hoefer Scientific Instruments, San Francisco, California, 1983. 8 A. Chrambach and D. Rodbard, Science 172, 440 (1971). 9 B. J. Davis, Ann. N.Y. Acad. Sci. 121, 404 (1964).
166
PREPARATION OF TOXINS
[23]
Preparative Gel Electrophoresis 1o A large preparative gel electrophoresis [Poly-Prep 100, Biachler Instruments Div., Nuclear-Chicago Corporation (Searle and Co.), Fort Lee, N J]. The advantage of this technique is that milligram amounts of highly purified toxin can be obtained directly from the crude ammonium sulfate prepared toxin. For example, from 250 mg of crude toxin (two runs), 11.3 mg of purified toxin A was obtained. No evidence for lipopolysaccharide contamination was found in biological studies. 11 In some preparations a toxic dose was at the level of 0.1 tzg Lowry protein. Toxin (80 to 150 mg) sample is dissolved in up to 15 ml of sample buffer (2.89 tzg Tris and 12.8 ml 1 M H3PO4 brought to 100 ml) and layered onto a preformed concentrating gel using riboflavin-polymerized acrylamide (see components and solutions, Table I) or by directly layering a toxin solution of Tris-glycine or Tris-phosphate sample buffer with 10% sucrose onto a 6-cm resolving gel. One drop of concentrated bromphenol blue is added as a marker dye. The column is continuously cooled at 6-8 °. Run time is approximately 36 hr in a Tris-glycine pH 8.9, 7.5% acrylamide running gel system with an elution flow rate of 20 ml/hr. Toxin bands are identified on analytical gels using 0.02 ml of a 2- to 4-ml collected fraction. A constant amperage is required, set at 50 mA. The voltage will rise slowly during the run from 250 to 400-450 V. The individual toxins are resolved into two peaks of approximately 20 consecutive 2-ml fractions, for example, with toxin B in fractions 58 to 70, and toxin A in fractions 135 to 155 (see analytical gel profile in Ref. 10). The toxin samples are pooled, dialyzed against 0.001 M KHzPO4, pH 7.0, buffer, and lyophilized. Storage of lyophilized material is at - 7 0 °, desiccated. Preparation of Toxin Subunit Components
Isolation of Biologically Active Subunits Using Sodium Dodecyl Sulfate (SDS)12,13 Although large-molecular-weight murine toxin is partially denatured by treatment with 0.5-1% SDS, it apparently retains partial activity even though dissociation occurs to a subunit of approximate MW 24,000. These subunits may be obtained by dissolving toxin protein in 1.0% SDS and incubating at 37 ° for 3 hr. Excess SDS is removed by dialysis against 0.1% ~0T. C. Montie and D. B. Montie, J. Bacteriol. 100, 535 (1969). 11 S. D. Brown and T. C. Montie, Infect. Imrnun. 18, 85 (1977). 12 T. C. Montie, D. B. Montie, S. A. Leon, C. A. Kennedy, and S. J, Ajl, Biochern. Biophys. Res. Commun. 33, 423 (1968). 13 T. C. Montie and D. B. Montie, Biochemistry 10, 2094 (1971).
Yersinia pestis PLAGUE TOXIN
[23]
167
TABLE I REQUIRED SOLUTIONS FOR BUCHLER POLY-PREP ACRYLAM1DE GEL ELECTROPHORESlS Buffers
Chemicals
Amount
Concentration (-fold)
Upper (per 2 liters) Lower (per 2 liters) Membrane (per liter) Sample (per 100 ml) Elution
Tris Glycine Tris 1 N HC1 Tris 1 N HCI 1 M H3PO4 Tris Lower buffer H20
12.64 g 7.88 g 72.6 ml 300 ml 48.4 g 200 ml 12.8 ml 2.85 g 1 part 2 parts
--3× 4× --1×
Stock solutions
Gel
Volume desired
Resolving
100 ml
Volume ratio 25 ml 25 ml
50 ml Concentrating ~
50 ml
30ml 15 ml
Additional solutions
15 ml 100 ml H20 100 ml H20
Component Acrylamide Bisacrylamide 1 N HC1 Tris TEMED Ammonium persulfate Acrylamide Bisacrylamide 1 M H3PO4 Tris TEMED Riboflavin Sucrose Bromphenol blue
Concentration /t00 ml 30.0 2.0 24.0 18.15 0.2- 0.4 0.15
g g ml g ml g
5.0 0.3- 1.25 12.8 2.85 0.1 2.0 40 5.0
g g ml g ml mg g mg
" This gel is hardened using a bank of fluorescent lights with riboflavin as the catalyst.
SDS for 17 to 24 hr. Under these conditions at least 60% of the toxic activity is retained." To avoid dialysis, passage through a Sephadex G-75 or G-100 column will remove excess SDS. The subunit form is retained and identified as the major retarded peak. In more dilute incubation solutions a polypeptide of 10,000-12,000 Da is obtained; however, the toxic activity of this unit may be eliminated. Specific conditions for subunit isolation and/or molecular weight determination include passing 1 to 2 mg/ml toxin in SDS-phosphate through a Sephadex G-100 column (1.5 × 90 cm) equilibrated with 0.1 M sodium phosphate buffer (pH 7.1).
168
PREPARATION OF TOXINS
[23]
Isolation of Inactivated Subunits with Organic Acids: Citric Acid and Acetic Acid Plus EDTA lz
Under these conditions lethal activity is lost, although attempts to reverse the acid denaturation have not been explored fully. In this method toxin (1-4 mg) is rapidly dissolved in 0.5 to 2.0 ml of citric acid (0.1 M, pH 2.2) and incubated at 37° for 1 hr or 26 ° for 24 hr (partial depolymerization). Complete depolymerization to a 12,000-Da fragment occurs after exposure at 37° for 24 hr. Alternatively, 0.1 M acetic acid with 10-3 M EDTA can be substituted for citric acid with approximately the same result. Toxin subunits are isolated by passing toxin dissolved in acid through a Sephadex G-50 column equilibrated with 0.001 M potassium phosphate (pH 7.0) and 0.1 M KC1. The high salt is added if column adsorption is a problem, but usually it can be omitted. Excess acid may be removed by passing the pooled two or three peak fractions through a second G-50 column. Acetic acid is the acid of choice for peptide determinations since radioactive citrate is bound to toxin even after tryptic digestion and separation of peptides by high-voltage electrophoresis. Isolation of Inactive Polypeptides by Gel Electrophoresis in a Phenol-Acetic Acid-Urea GeP °
Lyophilized native toxin fractions from the Btichler Poly-Prep gel electrophoresis are further separated into polypeptides by gel electrophoresis in a denaturing environment. Microgram amounts of toxin A or B (5 to 25 tzg) are dissolved in 0.1 to 0.2 ml phenol-acetic acid-water (2 : 1 : 0.5, v/v/v) and subjected to electrophoresis for 2 hr at room temperature through 7.5% (w/v) acrylamide gels containing 35% (v/v) acetic acid and 5 M urea. Protein bands are stained in the usual way with Amido black or Coomassie blue. Toxin A gives two bands: the least electropositive is designated No. 1 and the more electropositive band is designated No. 2. Toxin B exhibits two bands: a band corresponding to the No. 2 band of toxin A, and an additional No. 3 band of greater electrophoretic mobility than the No. 2 band. The No. 2 band (common to both toxins) always appears as the heaviest, in greatest proportion, or most densely stained band of the three. This method has been used by Rotten and Razin ~4 to identify mycoplasma membrane proteins and by Takayama et al.15 to distinguish mitochondrial membrane proteins.
~4 S. Rottem and S. Razin, J. Bacteriol. 94, 359 (1967). 15 K. Takayama, D. H. MacLennon, A. Tzagoloft, and C. D. Stoner, Arch. Biochem. Biophys. 114, 223 (1966).