Vol. 13, No. 3 Printed in U.S.A.
INFECTION AND IMMUNITY, Mar. 1976, p. 982-986 Copyright X) 1976 American Society for Microbiology
Purification of Staphylococcal Alpha-Toxin by Adsorption Chromatography on Glass PAUL CASSIDY AND SIDNEY HARSHMAN* Department of Microbiology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 Received for publication 5 November 1975
Staphylococcal alpha-toxin was purified from Staphylococcus aureus growth medium using adsorption chromatography on controlled pore glass beads. Elution of alpha-toxin from the unmodified glass surface of the beads with various anions generally followed the chaotropic series. Alpha-toxin, purified by glass bead chromatography, is composed of a single electrophoretic form, containing less than 2% of other forms. A recent review on staphylococcal alphatoxin (1) cites nine different purification procedures for the toxin. Since the review's publication, two other procedures have appeared (4, 10), one of which yields two forms of alphatoxin, each electrophoretically homogeneous under both native and denaturing conditions (10). The purification procedure described below has a number of advantages; it is a rapid two-step procedure which gives, in high yield, a protein of high specific activity usually containing less than 2% of other electrophoretic forms. Adsorption chromatography on glass beads is a method of protein purification whose use is at this point empirical. The technique has already proven to be highly successful in the purification of the mitochondrial enzyme f8-hydroxybutyrate dehydrogenase from crude mitochondrial extracts (3). Chromatography of substances on controlled pore glass beads has largely been restricted to permeation chromatography despite the known reversible adsorption of proteins and viruses to the beads (4). Adsorption has been prevented by treating the glass with polyethylene glycol (8) or manipulation of pH and electrolyte concentration (9). The reversible adsorption of proteins by unmodified glass surfaces is the basis of the alpha-toxin purification procedure which follows. (Preliminary reports of some of the work reported here were given at the 3rd International Symposium on Staphylococci and Staphylococcal Infections, 8-14 September 1975, Warsaw.) MATERIALS AND METHODS Staphylococcus aureus. S. aureus Woods strain 46 was identical to that used by Six and Harshman (10). It was grown in a medium containing the dialysate from yeast extract (2) and stored at -65 C in growth medium made 50% (vol/vol) with glycerol. All salts were Fisher reagent grade. The controlled
pore glass beads were product CPG-10-350 (75- to 125-,m particles) of Electro-Nucleonics, Fairfield, N. J. Diethylaminoethyl (DEAE)-Sephadex was supplied by Pharmacia Corporation. Hemolytic assay. The hemolytic assay is a serial twofold dilution of 50% hemolysis end point assay described by Bernheimer and Schwartz (2). Assay buffer used throughout contained 0.15 M NaCl, 0.02 M potassium phosphate, pH 7.4, and 1 mg of bovine serum albumin (PBSA) per ml. Aliquots of alpha-toxin were diluted to 2 ml with PBSA and 1 ml was taken for serial twofold dilution in PBSA. To 1 ml of diluted toxin, 1 ml of 1% (vol/vol) PBSAwashed rabbit erythrocytes was added and the dilution series was incubated at 37 C for 30 min. The tubes were then centrifuged for 5 min in a clinical centrifuge to remove cells and stroma, and the optical densities of the supernatants were determined at 545 nm. One hundred percent hemolysis was determined from a tube containing 0.1% (wt/ vol) sodium dodecyl sulfate. The tube representing 50% hemolysis was determined. One hemolytic unit is that amount of alpha-toxin producing 50% lysis in the above assay. The concentration of hemolytic units in a sample assayed is calculated from the known dilution factors. Usually, interpolation of values between adjacent assay tubes was done. The addition of 1 ml of 1% erythrocytes was included as a twofold dilution in the calculation. Rabbit erythrocytes were obtained by cardiac puncture and were stored in Alsever solution. Human erythrocytes were group O+ obtained as outdated blood from the Vanderbilt blood bank or occasionally from group O+ volunteers.
Analytical electrophoresis. Polyacrylamide gel electrophoresis was performed at pH 8.9 by the method of Davis (5) as modified by Six and Harshman (10). The gels were fixed and stained with Coomassie brillant blue by the method of Fairbanks (6). Crude toxin production. To prepare a starter culture, 100 ml of the medium referred to above was inoculated with S. aureus Woods strain 46 and grown for 24 h at 37 C. Ten milliliters of the starter culture was used to inoculate each of six 500-ml 982
PURIFICATION OF STAPHYLOCOCCAL ALPHA-TOXIN
VOL. 13, 1976
cultures, which were shaken at 37 C for 18 h. The cultures were then centrifuged at 30,000 x g for 30 min at 4 C to remove the organisms. The pooled supernatant was then used for alpha-toxin preparation. Preparation and cleaning of controlled pore glass beads. Controlled pore glass beads could be used directly for the alpha-toxin preparation after rinsing in distilled water and equilibration with 0.05 M potassium phosphate buffer, pH 6.8. After use, cleaning of the beads was performed by heating at 100 C in 15.7 N nitric acid on a steam bath for 2 h. On occasion, concentrated sulfuric acid containing sodium dichromate was also used. After extensive washing in distilled water, the beads were again ready for use. No decrease in capacity for alphatoxin adsorption was noted in beads recycled five times by this method. RESULTS
Purification procedure. The supernatant from the 3-liter culture (approximately 2,800 ml) was adjusted to pH 6.8 at 4 C by the addition of a small amount of 1 N HCl. The supernatant was then passed through a glass column (8 by 14 cm) containing 500 g of controlled pore glass beads which had been equilibrated with 0.05 M potassium phosphate buffer, pH 6.8. The column was then washed with five column volumes of the equilibration buffer. The toxin was eluted with 1 M potassium phosphate buffer, pH 7.5. The alpha-toxin activity routinely eluted in a 500- to 800-ml volume. In preliminary runs, the step elution described above was compared with a linear phosphate gradient elution at pH 7.5 from 0.05 to 1 M phosphate. The step elution procedure was chosen because it produced an alpha-toxin preparation of comparable specific activity to the gradient elution in a much shorter time period. The pooled alpha-toxin preparation eluted from the glass bead column appeared slightly yellow, presumably due to slight contamination by the brown pigments originally present in the yeast extract medium. Hemolytic activity re-
983
at this stage was usually 50 to 84% of the original activity (Table 1). When this material was analyzed by polyacrylamide gel electrophoresis at pH 8.9, it was found to contain a major basic protein band which coelectrophoresed with alpha-toxin form B in the nomenclature of Six and Harshman (10). An acidic protein contaminant representing approximately 5% of the total protein was also detected. The pooled alpha-toxin preparation was precipitated by the addition of ammonium sulfate to 90% saturation, and the precipitate was collected by centrifugation. The protein precipitate was dissolved in approximately 50 ml of 0.05 M potassium phosphate buffer, pH 8.5, and was then dialyzed versus 5 liters of the same buffer for 6 h. The dialyzed preparation was then passed through a column (4 by 20 cm) of DEAE-Sephadex which had been equilibrated with the same buffer. Under these conditions, alpha-toxin is not retained whereas the acidic protein contaminant is adsorbed. The alphatoxin preparation after DEAE-Sephadex appeared to be a single major protein band on polyacrylamide gel electrophoresis at pH 8.9 (Fig. 1) and was present as 98% form B, 2% form A. Overall recovery of hemolytic activity was approximately 80%. The extent of purification was difficult to determine accurately since the presence of large amounts of peptides, amino acids, and pigments in the original yeast extract medium interfered with all protein assays available. However, based on optical density at 280 nm, the overall purification of alphatoxin attained by the method was 800- to 1,000fold. The hemolytic activity ratio measured by performing hemolytic assays with rabbit and human erythrocytes revealed that the ratio dropped from 0.198 in the culture supernatant to 0.001 in the pooled activity from the glass bead column. The ratio of activities on the two types of erythrocytes is an indication of the relative contamination of the alpha-toxin preparation by staphylococcal delta-toxin (13). covery
TABLE 1. Purification of alpha-toxin on glass beads Total
Purification
TTotal TtlproproVol (ml)
ten (mg)
Total hemltcU
~~~~~~~Hemoytic Hmoytcieolti ytmc Hemolytic HemDmgol activ/g
UO20
ity
ra-
tio,
% Ac-
tivity recoyered
100 2.4 x 101 0.194 S. aureus growth medium 6.8 x 106 2,750 0.014 1.1 x 106 2,500 Effluent from glass beads 84 1 M K phosphate (pH 7.4) 223 5.68 x 106 2.56 x 104 2.82 x 104 0.001 726 elution 80 48r 0.001 5.44 x 10" 2.80 x 104 3.08 x 104 194 DEAE-Sephadex is El, nm = 11.0; OD28,,, Optical density at 280 nm. alpha-toxin Extinction coefficient of homogeneous Ratio of hemolytic activity on human and rabbit erythrocytes (human/rabbit). Volume after 90% saturated ammonium sulfate precipitation and passage through DEAE-Sephadex. r
984
CASSIDY AND HARSHMAN
I
INFECT. IMMUN.
potassium phosphate buffer, pH 7.4, at concentrations of 2 to 8 mg/ml. After dialysis against the same buffer, the protein solution was stored at -65 C. The hemolytic activity was stable for months under both storage conditions. As with alpha-toxin forms B and A prepared by preparative electrophoresis (10), alpha-toxin purified by the above method lost activity totally when lyophilized. Elution of purified alpha-toxin from glass beads by different anions. Alpha-toxin was eluted from small glass bead columns (Fig. 2) differentially by anions of the chaotropic series (7), and the potency of elution generally followed the series, as is true for the elution of f8hydroxybutyrate dehydrogenase under similar conditions (3). The two cations, choline and tris(hydroxymethyl)aminomethane in the chloride form, were also effective in eluting the toxin compared with KC1 or NaCl. Phosphate represented the only ion tested that deviated from the general order of elution potency. No toxin was eluted with 1 M solutions of any of the salts when the pH was maintained below pH 6.9.
2
FIG. 1. Polyacrylamide gel electrophoresis at pH 8.9 of alpha-toxin purified by adsorption on glass beads. Gel 1, 150 Mg of ammonium sulfate-precipitated protein from growth medium; gel 2, 15
pg
of
alpha-toxin after glass bead and DEAE-Sephadex chromatography.
Either the purified toxin was stored as a 90% saturated ammonium sulfate precipitate at 4 C or the precipitate was redissolved in 0.05 M
DISCUSSION The purification of staphylococcal alphatoxin by adsorption chromatography on glass beads offers a number of advantages over other methods. It is a procedure which is rapidly performed and can be completed in 2 to 3 days. The high-percentage recovery of hemolytic activity may be principally due to the time factor, since even homogeneous alpha-toxin seems to be subject to slow denaturation in dilute buffer. The protein is not exposed to extremes of pH, as is necessary during the preparative electrophoresis step in the purification scheme of Six and Harshman (10). During the electrophoresis step, the pH of the separating gel may reach 10.0. Exposure to such a pH extreme may have unknown modifying effects on the toxin and may be in part responsible for the tight binding of about 25 mol of tris(hydroxymethyl)aminomethane per mol of alpha-toxin (11). The electrophoretic form of alpha-toxin that is produced by purification on glass beads seems to be identical with the alpha-toxin form B previously described (10). Because alphatoxin form B and the slightly more acidic form A are recovered equally well from small glass bead columns (P. Cassidy, unpublished observation), alpha-toxin form A may have been generated from form B in the previously used lengthy purification procedure (10). The theoretical basis for the adsorption of proteins to unmodified glass surfaces is not well
VOL. 13, 1976
PURIFICATION OF STAPHYLOCOCCAL ALPHA-TOXIN NaSCN
KI
NONO3
985
Li Br
100 so
60 40 HE MOLYT IC ACT IVITY RECOVERED IN EACH FRACTION
20 0
aL
L
KCI
NOCI
00or
Choline CI
Tris Cl
K
Phosphale
Na Sulfate
Na Acetate
SOF 60O 40-
20
io
-J=
FIG. 2. Elution of alpha-toxin from small glass bead columns by different anions. Small glass bead columns (0.5 ml of beads in Pasteur pipettes) were prepared in 20 mM tris(hydroxymethyl)aminomethane(Tris)-chloride, pH 7.5. Alpha-toxin samples (50 Hig ofprotein in the same buffer) were applied to the columns and chromatography was performed at 4 C. After washing the columns with 3 ml of 0.1 M Tris-chloride, pH 7.5, during which no activity was eluted, the columns were eluted with 0.1 M Tris-chloride, pH 7.5, containing 1 M salt as listed in the figure. Three separate fractions were collected: 0.5, 1.0, and 2.0 ml. The percentage of the total activity recovered in each fraction is represented from left to right in each bar graph. Thus, with 1 M NaSCN, 20% of the total hemolytic activity is recovered in the first 0.5 ml of effluent, 56% in the next 1.0 ml, and none in the last 2.0 ml, giving a total recovery of 76%.
established. Controlled pore glass is 96% silica face than the overall charge on the toxin molewithout surface modification (4). The surface is cules. Titration of groups on the glass surface said to consist of hydroxyl groups and to display itself may also take place. a slight negative charge in aqueous media. In Hydrophobic interaction of alpha-toxin with addition to silica, the glass also contains boron the glass surface is indicated, since anions that in the form of B203, which represents 3 to 5% of are known to weaken hydrophobic protein-prothe content (14). The presence of a slight nega- tein and protein-lipid interactions are much tive charge on the glass surface may in part be more effective at toxin elution than anions responsible for the strong adsorption of basic known to strengthen such interactions (7). The proteins (4). An unknown portion of the glass so-called "chaotropic" series of anions is arsurface may have been dehydrated by heating ranged in order of decreasing potency in disorduring the preparation of the glass beads. The dering solvent water structure (7, 12). It is atheating process removes adjacent hydroxyl tractive to attribute adsorption of alpha-toxin groups and results in local areas of hydropho- to the glass surface to a hydrophobic interaction. Because alpha-toxin is a hemolytic toxin bicity (14). The adsorption and elution of alpha-toxin and apparently binds to erythrocyte memfrom glass beads may depend on at least three branes with high affinity, a local area rich in separate phenomena. Because the adsorption hydrophobic amino acids may exist on the surprocess is highly pH dependent, electrostatic face of the toxin molecules. A third phenomenon which may affect the interactions between the protein and the glass surface are obviously important. However, adsorption and elution of alpha-toxin from since adsorption of alpha-toxin does not occur at glass surfaces is the known structure-perturbpH values of 7.0 or above, it is hard to visualize ing potency of chaotropic ions (12). The stronger the adsorption process as ion exchange, since chaotropic ions such as SCN-, CI04-, and 1the isoelectric point for alpha-toxin form B is are known to inhibit the activity of a number of 8.4 (11). It may be that certain critical amino unrelated enzymes, and the 50% inhibition conacid residues that titrate at about pH 7.0 are centration generally is between 0.5 and 3 M more important for adsorption to the glass sur- (12). In other words, strong chaotropic ions may
986
INFECT. IMMUN.
CASSIDY AND HARSHMAN
reversibly denature protein molecules, causing vice grant RO1 AI-11564 National Institute of Allergy and Diseases and by National Science Foundation the molecule to assume a slightly altered ter- Infectious grant GB-38902. tiary structure. Such an altered molecule might Paul Cassidy is a Vanderbilt Biomedical Sciences Felno longer be able to maintain electrostatic or low. hydrophobic interactions with the glass surface LITERATURE CITED and would be eluted. The three possible phenomena offered here as explanations of adsorp1. Arbuthnott, J. P. 1970. Staphylococcal a-toxin, p. 189236. In S. J. Ajl, S. Kadis, and T. C. Montie (ed.), tion and elution of alpha-toxin from glass beads Microbial toxins, vol. 3. Academic Press Inc., New are obviously not mutually exclusive. The bindYork. may glass the toward a protein of ing behavior 2. Bernheimer, A. W., and L. L. Schwartz. 1963. Isolation be the result of these three phenomena and and composition of staphylococcal a-toxin. J. Gen. Microbiol. 30:455-468. others in combination. H., and S. Fleischer. 1974. Purification of D-,8Phosphate was unexpectedly found to elute 3. Bock, hydroxybutyrate apodehydrogenase, a lecithin-realpha-toxin from glass beads. Since the presquiring enzyme, p. 374-391. In S. Fleischer, and L. ence of phosphate usually strengthens hydroPacker (ed.), Methods in enzymology, vol. 31. Academic Press Inc., New York. phobic interactions and it is the only exception controlled-pore glass operation instructions. Elecin the elution order for alpha-toxin, it may 4. CPG tro-Nucleonics, Inc., Fairfield, N. J. cause protein elution through direct binding to 5. Davis, B. J. 1964. Disc electrophoresis-II, method and the protein itself. Since phosphate under simiapplication to human serum proteins. Ann. N. Y. Acad. Sci. 121: 404-427. lar conditions does not cause the elution of /8G., T. L. Steck, and D. Wallach. 1971. hydroxybutyrate dehydrogenase from glass 6. Fairbanks, Electrophoretic analysis of the major polypeptides of beads (3), phosphate elution of protein from the human erythrocyte membrane. Biochemistry 10: glass will probably prove not to be a general 2606-2624. phenomenon. Phosphate was chosen to elute 7. Hatefi, Y., and W. Hanstein. 1974. Destabilization of membranes with chaotropic ions, p. 770-790. In S. alpha-toxin in the preparation procedure beFleischer and L. Packer (ed.), Methods in enzymolcause it generally stabilizes proteins in solution ogy, vol. 31. Academic Press Inc., New York. (12). Also, exposure of alpha-toxin to high con8. Hawk, G. L., J. A. Cameron, and L. B. Dufault. 1972. Chromatography of biological materials on polyethylcentrations of halide ions was avoided because ene glycol treated controlled pore glass. Prep. Biowe have found that iodination of the toxin is chem. 2: 193-203. possible and leads to inactivation of hemolytic 9. Marcinka, K. 1972. Application of permeation chromaactivity. tography on controlled-pore glass in the purification of plant viruses. Acta Virol. 16:53-62. Adsorption chromatography on glass beads H. R., and S. Harshman. 1973. Purification and may have general application in the purifica- 10. Six,properties of two forms of staphylococcal a-toxin. Biotion of other bacterial exoproteins. Delta-toxin chemistry 12:2672-2677. activity is also adsorbed from A. aureus growth 11. Six, H. R., and S. Harshman. 1973. Physical and chemical studies on staphylococcal a-toxins A and B. Biomedium as can be seen by a comparison of the 12:2677-2683. hemolytic activity ratios (hemolytic activity as- 12. vonchemistry Hippel, P. H., and T. Scheich. 1969. The effects of sayed on human cells versus rabbit cells) of the neutral salts on the structure and conformational growth medium and the effluent from glass stability of biological molecules, p. 417-574. In S. N. Timasheff and G. Fasman (ed.), Marcel Dekker, beads (Table 1). Chromatography on glass New York. beads should be explored for its possible useful- 13. Wadstrom, T., and R. Mo6lby. 1972. Some biological ness in the purification of delta-toxin and other properties of purified staphylococcal haemolysin.
bacterial exoproteins.
ACKNOWLEDGMENTS This work was supported in part by Public Health Ser-
Toxicon 10:511-519. 14. Watanabe, M., and I. Kato. 1974. Purification and some properties of staphylococcal a-toxin. Jpn. J. Exp. Med. 44:165-178.
INFECTION AND IMMUNITY, Mar. 1976, p. 982-986 Copyright X) 1976 American Society for Microbiology
Purification of Staphylococcal Alpha-Toxin by Adsorption Chromatography on Glass PAUL CASSIDY AND SIDNEY HARSHMAN* Department of Microbiology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 Received for publication 5 November 1975
Staphylococcal alpha-toxin was purified from Staphylococcus aureus growth medium using adsorption chromatography on controlled pore glass beads. Elution of alpha-toxin from the unmodified glass surface of the beads with various anions generally followed the chaotropic series. Alpha-toxin, purified by glass bead chromatography, is composed of a single electrophoretic form, containing less than 2% of other forms. A recent review on staphylococcal alphatoxin (1) cites nine different purification procedures for the toxin. Since the review's publication, two other procedures have appeared (4, 10), one of which yields two forms of alphatoxin, each electrophoretically homogeneous under both native and denaturing conditions (10). The purification procedure described below has a number of advantages; it is a rapid two-step procedure which gives, in high yield, a protein of high specific activity usually containing less than 2% of other electrophoretic forms. Adsorption chromatography on glass beads is a method of protein purification whose use is at this point empirical. The technique has already proven to be highly successful in the purification of the mitochondrial enzyme f8-hydroxybutyrate dehydrogenase from crude mitochondrial extracts (3). Chromatography of substances on controlled pore glass beads has largely been restricted to permeation chromatography despite the known reversible adsorption of proteins and viruses to the beads (4). Adsorption has been prevented by treating the glass with polyethylene glycol (8) or manipulation of pH and electrolyte concentration (9). The reversible adsorption of proteins by unmodified glass surfaces is the basis of the alpha-toxin purification procedure which follows. (Preliminary reports of some of the work reported here were given at the 3rd International Symposium on Staphylococci and Staphylococcal Infections, 8-14 September 1975, Warsaw.) MATERIALS AND METHODS Staphylococcus aureus. S. aureus Woods strain 46 was identical to that used by Six and Harshman (10). It was grown in a medium containing the dialysate from yeast extract (2) and stored at -65 C in growth medium made 50% (vol/vol) with glycerol. All salts were Fisher reagent grade. The controlled
pore glass beads were product CPG-10-350 (75- to 125-,m particles) of Electro-Nucleonics, Fairfield, N. J. Diethylaminoethyl (DEAE)-Sephadex was supplied by Pharmacia Corporation. Hemolytic assay. The hemolytic assay is a serial twofold dilution of 50% hemolysis end point assay described by Bernheimer and Schwartz (2). Assay buffer used throughout contained 0.15 M NaCl, 0.02 M potassium phosphate, pH 7.4, and 1 mg of bovine serum albumin (PBSA) per ml. Aliquots of alpha-toxin were diluted to 2 ml with PBSA and 1 ml was taken for serial twofold dilution in PBSA. To 1 ml of diluted toxin, 1 ml of 1% (vol/vol) PBSAwashed rabbit erythrocytes was added and the dilution series was incubated at 37 C for 30 min. The tubes were then centrifuged for 5 min in a clinical centrifuge to remove cells and stroma, and the optical densities of the supernatants were determined at 545 nm. One hundred percent hemolysis was determined from a tube containing 0.1% (wt/ vol) sodium dodecyl sulfate. The tube representing 50% hemolysis was determined. One hemolytic unit is that amount of alpha-toxin producing 50% lysis in the above assay. The concentration of hemolytic units in a sample assayed is calculated from the known dilution factors. Usually, interpolation of values between adjacent assay tubes was done. The addition of 1 ml of 1% erythrocytes was included as a twofold dilution in the calculation. Rabbit erythrocytes were obtained by cardiac puncture and were stored in Alsever solution. Human erythrocytes were group O+ obtained as outdated blood from the Vanderbilt blood bank or occasionally from group O+ volunteers.
Analytical electrophoresis. Polyacrylamide gel electrophoresis was performed at pH 8.9 by the method of Davis (5) as modified by Six and Harshman (10). The gels were fixed and stained with Coomassie brillant blue by the method of Fairbanks (6). Crude toxin production. To prepare a starter culture, 100 ml of the medium referred to above was inoculated with S. aureus Woods strain 46 and grown for 24 h at 37 C. Ten milliliters of the starter culture was used to inoculate each of six 500-ml 982
PURIFICATION OF STAPHYLOCOCCAL ALPHA-TOXIN
VOL. 13, 1976
cultures, which were shaken at 37 C for 18 h. The cultures were then centrifuged at 30,000 x g for 30 min at 4 C to remove the organisms. The pooled supernatant was then used for alpha-toxin preparation. Preparation and cleaning of controlled pore glass beads. Controlled pore glass beads could be used directly for the alpha-toxin preparation after rinsing in distilled water and equilibration with 0.05 M potassium phosphate buffer, pH 6.8. After use, cleaning of the beads was performed by heating at 100 C in 15.7 N nitric acid on a steam bath for 2 h. On occasion, concentrated sulfuric acid containing sodium dichromate was also used. After extensive washing in distilled water, the beads were again ready for use. No decrease in capacity for alphatoxin adsorption was noted in beads recycled five times by this method. RESULTS
Purification procedure. The supernatant from the 3-liter culture (approximately 2,800 ml) was adjusted to pH 6.8 at 4 C by the addition of a small amount of 1 N HCl. The supernatant was then passed through a glass column (8 by 14 cm) containing 500 g of controlled pore glass beads which had been equilibrated with 0.05 M potassium phosphate buffer, pH 6.8. The column was then washed with five column volumes of the equilibration buffer. The toxin was eluted with 1 M potassium phosphate buffer, pH 7.5. The alpha-toxin activity routinely eluted in a 500- to 800-ml volume. In preliminary runs, the step elution described above was compared with a linear phosphate gradient elution at pH 7.5 from 0.05 to 1 M phosphate. The step elution procedure was chosen because it produced an alpha-toxin preparation of comparable specific activity to the gradient elution in a much shorter time period. The pooled alpha-toxin preparation eluted from the glass bead column appeared slightly yellow, presumably due to slight contamination by the brown pigments originally present in the yeast extract medium. Hemolytic activity re-
983
at this stage was usually 50 to 84% of the original activity (Table 1). When this material was analyzed by polyacrylamide gel electrophoresis at pH 8.9, it was found to contain a major basic protein band which coelectrophoresed with alpha-toxin form B in the nomenclature of Six and Harshman (10). An acidic protein contaminant representing approximately 5% of the total protein was also detected. The pooled alpha-toxin preparation was precipitated by the addition of ammonium sulfate to 90% saturation, and the precipitate was collected by centrifugation. The protein precipitate was dissolved in approximately 50 ml of 0.05 M potassium phosphate buffer, pH 8.5, and was then dialyzed versus 5 liters of the same buffer for 6 h. The dialyzed preparation was then passed through a column (4 by 20 cm) of DEAE-Sephadex which had been equilibrated with the same buffer. Under these conditions, alpha-toxin is not retained whereas the acidic protein contaminant is adsorbed. The alphatoxin preparation after DEAE-Sephadex appeared to be a single major protein band on polyacrylamide gel electrophoresis at pH 8.9 (Fig. 1) and was present as 98% form B, 2% form A. Overall recovery of hemolytic activity was approximately 80%. The extent of purification was difficult to determine accurately since the presence of large amounts of peptides, amino acids, and pigments in the original yeast extract medium interfered with all protein assays available. However, based on optical density at 280 nm, the overall purification of alphatoxin attained by the method was 800- to 1,000fold. The hemolytic activity ratio measured by performing hemolytic assays with rabbit and human erythrocytes revealed that the ratio dropped from 0.198 in the culture supernatant to 0.001 in the pooled activity from the glass bead column. The ratio of activities on the two types of erythrocytes is an indication of the relative contamination of the alpha-toxin preparation by staphylococcal delta-toxin (13). covery
TABLE 1. Purification of alpha-toxin on glass beads Total
Purification
TTotal TtlproproVol (ml)
ten (mg)
Total hemltcU
~~~~~~~Hemoytic Hmoytcieolti ytmc Hemolytic HemDmgol activ/g
UO20
ity
ra-
tio,
% Ac-
tivity recoyered
100 2.4 x 101 0.194 S. aureus growth medium 6.8 x 106 2,750 0.014 1.1 x 106 2,500 Effluent from glass beads 84 1 M K phosphate (pH 7.4) 223 5.68 x 106 2.56 x 104 2.82 x 104 0.001 726 elution 80 48r 0.001 5.44 x 10" 2.80 x 104 3.08 x 104 194 DEAE-Sephadex is El, nm = 11.0; OD28,,, Optical density at 280 nm. alpha-toxin Extinction coefficient of homogeneous Ratio of hemolytic activity on human and rabbit erythrocytes (human/rabbit). Volume after 90% saturated ammonium sulfate precipitation and passage through DEAE-Sephadex. r
984
CASSIDY AND HARSHMAN
I
INFECT. IMMUN.
potassium phosphate buffer, pH 7.4, at concentrations of 2 to 8 mg/ml. After dialysis against the same buffer, the protein solution was stored at -65 C. The hemolytic activity was stable for months under both storage conditions. As with alpha-toxin forms B and A prepared by preparative electrophoresis (10), alpha-toxin purified by the above method lost activity totally when lyophilized. Elution of purified alpha-toxin from glass beads by different anions. Alpha-toxin was eluted from small glass bead columns (Fig. 2) differentially by anions of the chaotropic series (7), and the potency of elution generally followed the series, as is true for the elution of f8hydroxybutyrate dehydrogenase under similar conditions (3). The two cations, choline and tris(hydroxymethyl)aminomethane in the chloride form, were also effective in eluting the toxin compared with KC1 or NaCl. Phosphate represented the only ion tested that deviated from the general order of elution potency. No toxin was eluted with 1 M solutions of any of the salts when the pH was maintained below pH 6.9.
2
FIG. 1. Polyacrylamide gel electrophoresis at pH 8.9 of alpha-toxin purified by adsorption on glass beads. Gel 1, 150 Mg of ammonium sulfate-precipitated protein from growth medium; gel 2, 15
pg
of
alpha-toxin after glass bead and DEAE-Sephadex chromatography.
Either the purified toxin was stored as a 90% saturated ammonium sulfate precipitate at 4 C or the precipitate was redissolved in 0.05 M
DISCUSSION The purification of staphylococcal alphatoxin by adsorption chromatography on glass beads offers a number of advantages over other methods. It is a procedure which is rapidly performed and can be completed in 2 to 3 days. The high-percentage recovery of hemolytic activity may be principally due to the time factor, since even homogeneous alpha-toxin seems to be subject to slow denaturation in dilute buffer. The protein is not exposed to extremes of pH, as is necessary during the preparative electrophoresis step in the purification scheme of Six and Harshman (10). During the electrophoresis step, the pH of the separating gel may reach 10.0. Exposure to such a pH extreme may have unknown modifying effects on the toxin and may be in part responsible for the tight binding of about 25 mol of tris(hydroxymethyl)aminomethane per mol of alpha-toxin (11). The electrophoretic form of alpha-toxin that is produced by purification on glass beads seems to be identical with the alpha-toxin form B previously described (10). Because alphatoxin form B and the slightly more acidic form A are recovered equally well from small glass bead columns (P. Cassidy, unpublished observation), alpha-toxin form A may have been generated from form B in the previously used lengthy purification procedure (10). The theoretical basis for the adsorption of proteins to unmodified glass surfaces is not well
VOL. 13, 1976
PURIFICATION OF STAPHYLOCOCCAL ALPHA-TOXIN NaSCN
KI
NONO3
985
Li Br
100 so
60 40 HE MOLYT IC ACT IVITY RECOVERED IN EACH FRACTION
20 0
aL
L
KCI
NOCI
00or
Choline CI
Tris Cl
K
Phosphale
Na Sulfate
Na Acetate
SOF 60O 40-
20
io
-J=
FIG. 2. Elution of alpha-toxin from small glass bead columns by different anions. Small glass bead columns (0.5 ml of beads in Pasteur pipettes) were prepared in 20 mM tris(hydroxymethyl)aminomethane(Tris)-chloride, pH 7.5. Alpha-toxin samples (50 Hig ofprotein in the same buffer) were applied to the columns and chromatography was performed at 4 C. After washing the columns with 3 ml of 0.1 M Tris-chloride, pH 7.5, during which no activity was eluted, the columns were eluted with 0.1 M Tris-chloride, pH 7.5, containing 1 M salt as listed in the figure. Three separate fractions were collected: 0.5, 1.0, and 2.0 ml. The percentage of the total activity recovered in each fraction is represented from left to right in each bar graph. Thus, with 1 M NaSCN, 20% of the total hemolytic activity is recovered in the first 0.5 ml of effluent, 56% in the next 1.0 ml, and none in the last 2.0 ml, giving a total recovery of 76%.
established. Controlled pore glass is 96% silica face than the overall charge on the toxin molewithout surface modification (4). The surface is cules. Titration of groups on the glass surface said to consist of hydroxyl groups and to display itself may also take place. a slight negative charge in aqueous media. In Hydrophobic interaction of alpha-toxin with addition to silica, the glass also contains boron the glass surface is indicated, since anions that in the form of B203, which represents 3 to 5% of are known to weaken hydrophobic protein-prothe content (14). The presence of a slight nega- tein and protein-lipid interactions are much tive charge on the glass surface may in part be more effective at toxin elution than anions responsible for the strong adsorption of basic known to strengthen such interactions (7). The proteins (4). An unknown portion of the glass so-called "chaotropic" series of anions is arsurface may have been dehydrated by heating ranged in order of decreasing potency in disorduring the preparation of the glass beads. The dering solvent water structure (7, 12). It is atheating process removes adjacent hydroxyl tractive to attribute adsorption of alpha-toxin groups and results in local areas of hydropho- to the glass surface to a hydrophobic interaction. Because alpha-toxin is a hemolytic toxin bicity (14). The adsorption and elution of alpha-toxin and apparently binds to erythrocyte memfrom glass beads may depend on at least three branes with high affinity, a local area rich in separate phenomena. Because the adsorption hydrophobic amino acids may exist on the surprocess is highly pH dependent, electrostatic face of the toxin molecules. A third phenomenon which may affect the interactions between the protein and the glass surface are obviously important. However, adsorption and elution of alpha-toxin from since adsorption of alpha-toxin does not occur at glass surfaces is the known structure-perturbpH values of 7.0 or above, it is hard to visualize ing potency of chaotropic ions (12). The stronger the adsorption process as ion exchange, since chaotropic ions such as SCN-, CI04-, and 1the isoelectric point for alpha-toxin form B is are known to inhibit the activity of a number of 8.4 (11). It may be that certain critical amino unrelated enzymes, and the 50% inhibition conacid residues that titrate at about pH 7.0 are centration generally is between 0.5 and 3 M more important for adsorption to the glass sur- (12). In other words, strong chaotropic ions may
986
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CASSIDY AND HARSHMAN
reversibly denature protein molecules, causing vice grant RO1 AI-11564 National Institute of Allergy and Diseases and by National Science Foundation the molecule to assume a slightly altered ter- Infectious grant GB-38902. tiary structure. Such an altered molecule might Paul Cassidy is a Vanderbilt Biomedical Sciences Felno longer be able to maintain electrostatic or low. hydrophobic interactions with the glass surface LITERATURE CITED and would be eluted. The three possible phenomena offered here as explanations of adsorp1. Arbuthnott, J. P. 1970. Staphylococcal a-toxin, p. 189236. In S. J. Ajl, S. Kadis, and T. C. Montie (ed.), tion and elution of alpha-toxin from glass beads Microbial toxins, vol. 3. Academic Press Inc., New are obviously not mutually exclusive. The bindYork. may glass the toward a protein of ing behavior 2. Bernheimer, A. W., and L. L. Schwartz. 1963. Isolation be the result of these three phenomena and and composition of staphylococcal a-toxin. J. Gen. Microbiol. 30:455-468. others in combination. H., and S. Fleischer. 1974. Purification of D-,8Phosphate was unexpectedly found to elute 3. Bock, hydroxybutyrate apodehydrogenase, a lecithin-realpha-toxin from glass beads. Since the presquiring enzyme, p. 374-391. In S. Fleischer, and L. ence of phosphate usually strengthens hydroPacker (ed.), Methods in enzymology, vol. 31. Academic Press Inc., New York. phobic interactions and it is the only exception controlled-pore glass operation instructions. Elecin the elution order for alpha-toxin, it may 4. CPG tro-Nucleonics, Inc., Fairfield, N. J. cause protein elution through direct binding to 5. Davis, B. J. 1964. Disc electrophoresis-II, method and the protein itself. Since phosphate under simiapplication to human serum proteins. Ann. N. Y. Acad. Sci. 121: 404-427. lar conditions does not cause the elution of /8G., T. L. Steck, and D. Wallach. 1971. hydroxybutyrate dehydrogenase from glass 6. Fairbanks, Electrophoretic analysis of the major polypeptides of beads (3), phosphate elution of protein from the human erythrocyte membrane. Biochemistry 10: glass will probably prove not to be a general 2606-2624. phenomenon. Phosphate was chosen to elute 7. Hatefi, Y., and W. Hanstein. 1974. Destabilization of membranes with chaotropic ions, p. 770-790. In S. alpha-toxin in the preparation procedure beFleischer and L. Packer (ed.), Methods in enzymolcause it generally stabilizes proteins in solution ogy, vol. 31. Academic Press Inc., New York. (12). Also, exposure of alpha-toxin to high con8. Hawk, G. L., J. A. Cameron, and L. B. Dufault. 1972. Chromatography of biological materials on polyethylcentrations of halide ions was avoided because ene glycol treated controlled pore glass. Prep. Biowe have found that iodination of the toxin is chem. 2: 193-203. possible and leads to inactivation of hemolytic 9. Marcinka, K. 1972. Application of permeation chromaactivity. tography on controlled-pore glass in the purification of plant viruses. Acta Virol. 16:53-62. Adsorption chromatography on glass beads H. R., and S. Harshman. 1973. Purification and may have general application in the purifica- 10. Six,properties of two forms of staphylococcal a-toxin. Biotion of other bacterial exoproteins. Delta-toxin chemistry 12:2672-2677. activity is also adsorbed from A. aureus growth 11. Six, H. R., and S. Harshman. 1973. Physical and chemical studies on staphylococcal a-toxins A and B. Biomedium as can be seen by a comparison of the 12:2677-2683. hemolytic activity ratios (hemolytic activity as- 12. vonchemistry Hippel, P. H., and T. Scheich. 1969. The effects of sayed on human cells versus rabbit cells) of the neutral salts on the structure and conformational growth medium and the effluent from glass stability of biological molecules, p. 417-574. In S. N. Timasheff and G. Fasman (ed.), Marcel Dekker, beads (Table 1). Chromatography on glass New York. beads should be explored for its possible useful- 13. Wadstrom, T., and R. Mo6lby. 1972. Some biological ness in the purification of delta-toxin and other properties of purified staphylococcal haemolysin.
bacterial exoproteins.
ACKNOWLEDGMENTS This work was supported in part by Public Health Ser-
Toxicon 10:511-519. 14. Watanabe, M., and I. Kato. 1974. Purification and some properties of staphylococcal a-toxin. Jpn. J. Exp. Med. 44:165-178.
