Int. J. Biochem. Vol. 24,No. 7,pp.1073-1079, 1992 Printed in Great Britain. All rights reserved

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PURIFICATION AND CHARACTERIZATION OF STREPTOLYSIN 0 FROM S7’REPTOCOCCUS PYOGENES FRANCEXA CANALIAS, JORDI VWER, JORGE BELETA,FRANCE% GONZALEZ-SASTRE and F. JAVIER GELLA Departament de Bioquimica i Biologia Molecular, Universitat Autonoma de Barcelona, Hospital de la Santa Creu i Sant Pau, Sant Antoni Maria Claret 167, 08025 Barcelona, Spain [Tel. 456 19 00 ext. 45; Fnx 347 81 601 (Receitred 24 October 1991) Abstract-l. Streptolysin

0, an exotoxin produced by group A j?-hemolytic streptococci, has been purified from Streptococcus pyogenes culture supernatants. 2. The isolation and purification procedure involved ammonium sulphate and polyethylene glycol precipitations, and ion-exchange chromatographies on CM-Sepharose and Mono Q. 3. The proposed procedure introduces two ion-exchange chromatography steps making the purification process simpler and, especially, more reproducible than other described protocols. 4. The purified streptolysin 0 was hemolytically active, had a specific activity of 415,000 HU/mg, an optimum pH of 7.0, a relative molecular mass of 60,100 and an isoelectric pH of 7.5.

INTRODUCTION Streptolysin

0 is a toxic immunogenic

protein

pro-

duced by most strains of group A and many strains of groups C and G streptococci, particularly those causing human infections (Todd, 1932). It causes cytolysis of some eukaryotic cells, especially erythrocytes, from mammals and other species. A characteristic of the toxin is its sensitivity to oxidation in the presence of oxygen or oxidizing agents. Its biological activities are lost by oxidation but can be restored by reduction of the toxin with thiols (Alouf, 1980). Herbert and Todd (1941) proposed that streptolysin 0 is a protein containing at least one disulphide bond in the oxidized state (biologically inactive) which is reduced into free SH groups in the biologically active state. The hemolytic and other toxic effects of streptolysin 0 are also suppressed by small amounts of cholesterol and other sterols possessing a particular configuration (Hewitt et al., 1939). Streptolysin 0 is the prototype of sulphydrylactivated cytolysins elaborated by gram-positive bacteria. These toxins have several common properties (Bemheimer, 1974, 1976; Cowell and Bernheimer, 1977): they show maximal activity in the presence of reducing agents containing thiol groups, their activities are inhibited by cholesterol, they compete for a common membrane binding site which is believed to be cholesterol, they have similar molecular weights and similar isoelectric points, and their hemolytic activity is neutralized by antistreptolysin 0 and by antitetanolysin. The purified streptolysin 0 is used in clinical laboratories for the determination of antistreptolysin 0. A high proportion of patients with group A

streptococcal infection develop specific antibodies to streptolysin 0 which can combine specifically with the toxin inhibiting its hemolytic activity (Todd, 1932). Several authors have described the purification and characterization of streptolysin 0 present in culture supernatants of Streptococcus pyogenes (Herbert, 1941; Pentz and Shigemura, 1955; Alouf and Raynard, 1962, 1967, 1973; Van Epps and Anderson, 1969; Prigent et al., 1978; Linder, 1979; Gazzei et al., 1982; Bhakdi et al., 1984; Shany et af., 1973). These procedures combine several conventional methods including salting-out, ion-exchange chromatography, gel filtration, isoelectric focusing and preparative acrylamide gel electrophoresis. The isolation of the toxin poses serious problems due to the multiplicity of extracellular components in the crude material (NAD-glycohydrolase, streptokinase, DNAhydrolases or proteases), the low amount of streptolysin 0 in the medium and its tendency to irreversible denaturation. Most of the investigators place the molecular weight of streptolysin 0 in the range of 55,000-75,000 (Alouf, 1980; Van Epps and Andersen, 1969; Linder, 1979; Shany et al., 1973) and the isoelectric point is described to be between 6.5 and 7.5 (Alouf and Raynaud, 1973; Bhakdi et al., 1984; Shany et al., 1973; Smyth and Fehrenbach, 1974; Suzuki et al., 1988). The possible presence of two distinct forms of the toxin has been accounted by Alouf and Raynaud (1973) and more recently by Bhakdi et al. (1984). We describe here the purification of streptolysin 0 from bacterial culture supernatants by a novel and highly reproducible procedure. The described process is simpler and gives more purified streptolysin 0 than previously published methods.

1073

1074

FRANCESCACANALIASet al. Table 1. Summary

of the fractionation

Fraction Culture

supernatant

of culture supernatant different pH

Protein (mg)

Act. (kHW

Yield (%)

with ammonium sp. act. (kHU/mn)

sulphate Purification (fold)

30600

1364

100

0.044

I

pp 55% pH 6.5 pp 80% pH 6.5 sn 80% pH 6.5

590 I754 2366

420 564 100

31 41

0.712 0.321 -

I6 7

pp pp pp pp

II7 314 1100 1196 1875

31 580 1275 100

2 42 93

1.85 1.16

42 26

120 850 x91 I857

156 I857 90 60

II 136 7

40% pH 7.4 50% pH 7.4 65% pH 7.4 85% pH 7.4

sn 85% pH 7.4 40% pH X.0 pp 60% pH 8.0 pp 75% pH 8.0 pp

sn 75% DH 8.0 Initial

supernatant

MATERIALS

volume

was I

Materials

Polyetylene glycol 6000 was from Baker Chemicals, Deventer, Holland; CM-Sepharose-CL-6B, Sephacryl S-200 and Mono Q HR 5/5 anion-exchange column were from Pharmacia LKB Biotechnology, Uppsala, Sweden. Coomassie Brilliant Blue and electrophoresis reagents were from Bio-Rad, Richmond, Calif.. U.S.A. Isoelectric focusing reagents were from Pharmacia LKB Biotechnology, Uppsala, Sweden. Reagents for the culture of Sfreptococcus pyogenes were from Difco Laboratories, Detroit, Mich.. U.S.A. Group A type 3 Streptococcus pyogenes strain 10389 was obtained from the American Type Culture Collection. Reagents for NAD-glycohydrolase determination were from Boehringer Mannheim GmbH, Fed. Rep. Germany. Reagents for DNA-hydrolase determination were from Wampole Laboratories, Cranbury, NJ., U.S.A. Streptococcus pyogenes culture

A freeze-dried ampoule of Streptococcus pyogenes was reconstituted in sterile water, sowed on 5% sheep blood agar plates, and incubated at 37°C for 24 hr. Colonies surrounded by zones of hemolysis were subcultured into 3 I of 37 g/l Brain Heart Infusion broth containing 20 g/l yeast extract and 50 g/l glucose, and incubated at 37°C for 8 hr. This product was inoculated in 30 I of the same culture medium and incubated at 37°C for 16 hr. The pH of the culture was adjusted to 7.0 with IO mol/l NaOH. At the end of culture the microorganisms were inactivated by addition of 0.1 g/l mertiolate and sedimented by centrifugation. Assay of streptoiysin 0

Streptolysin 0 was measured as described by Herbert (1941) with some modifications. The sample to be tested was serially diluted in 36 mmol/l phosphate buffer pH 7.0 containing 126 mmol/l sodium chloride and 1 g/l bovine serum albumin. 0.5 ml of 150 mmol/l 2-mercaptoethanol was added to 1ml of each dilution of the sample and the mixture was incubated at 30°C for 15 min. Each reaction mixture was then supplemented with 0.5 ml of a rabbit erythrocyte of the fractionation

supernatant

pp AS 65% pH 7.4 pp 20% pH 8.0 pp 30% pH 8.0 sn 30% DH 8.0 Initial supernatant

29 49

suspension (1.58 x IO8cell/ml) in the same buffer and incubated at 30°C for 20min. At the end of the incubation, reaction mixtures were chilled with ice-water and centrifuged. Hemoglobin concentration in the supematants was determined spectrophotometrically at 541 nm. Blanks without sample and controls of total hemolysis were also run. The hemolysis of each sample was calculated by using the following equation: % Hemolysis =

A sample - A blank A total hemolysis - A blank

Determination of protein

Protein was quantified using the method described by Bradford (1976) with Coomassie Brilliant Blue as reagent and bovine serum albumin as standard. Protein was also quantified spectrophotometrically at 280 nm. Electrophoresis on polyacrylamide gels

Polyacrylamide gels electrophoresis in the presence of sodium dodecyl sulphate using a discontinuous buffer system was performed according to Laemmli (1970) and Laemmli and Favre (1973). Gels were stained with Coomassie Brilliant Blue R 250. Isoelectric focusing on polyacrylamide gels

Isoelectric focusing was carried out on polyacrylamide gels containing carrier ampholytes in the pH range from 3.5 to 9.5. The anode contained 1 mol/l phosphoric acid and the cathode 1 mol/l NaOH. After isoelectric focusing polyacrylamide plates were cut in two parts. One was stained with Coomassie Brilliant Blue R 250, and the other was cut in 2 mm slices which were extracted in 36 mmol/l phosphate buffer pH 7.0 containing 126 mmol/l sodium chloride and 1 g/l bovine serum albumin. The extraction fluids obtained with polyethylene

glycol 6000

Yield (%)

30600

I364

100

0.044

I

II00

1275

93

I.160

26

1027 5 -

75 -

6.300 -

143 -

volume was I I (AS: ammonium

Sp. act. (kHU/mg)

Purification (fold)

Act. (kHU)

163 134 588

X 100

One hemolytic unit (HU) was defined as that amount of streptolysin 0 which cause hemolysis of 50% of the erythrocytes (approx 3.95 x IO’) under the standard assay conditions described. Hemolytic activity concentration was calculated by interpolation of percentage of hemolysis in a calibration plot.

of culture supernatant

Protein (mg)

Fraction Culture

1.30 2.18

I (pp: precipitate; sn: supernatant)

AND METHODS

Table 2. Summary

at

sulphate; pp: precipitate;

sn: supernatant).

Streptolysin 0 purification

1075 03

0 Protem A260 0

0 Protein A260nm

“m

0 NoCl (mol/L)

NaCl imol/Ll

A SLO A541 nm

06

; u

02

2 2

g

008

E G

g

n

B a

01 004 I

P

Fraction number

Fig. I. Ion-exchange chromatography on CM-SepharoseCL-6B. Concentrate from the culture supernatant was applied to the column and the chromatography was run as described in the text. The collected fractions were assayed for conductivity (NaCI mol/l), absorbance at 280 nm (protein) and absorbance at 541 nm (streptolysin 0 activity).

after stirring activity.

overnight

at 4‘C were assayed

for hemolytic

RESULTS

Preparation of a concentrate from the culture supernatant

Culture supernatant obtained as described in Materials and Methods was concentrated in order to avoid the handling of high volumes of material. Fractional precipitations of proteins with salts and polymers were tested in this concentration step. Solid ammonium sulphate was added to the culture supernatant at different concentrations. The supernatant was previously adjusted to different pH values by dropwise addition of 1 mol/l acetic acid or solid Tris. After 30 min at 4°C the precipitates were collected by centrifugation at 10,000 g for 20 min, and 70

(

I 50

60 40

Froctlon number

Fig. 3. Ion-exchange chromatography on Mono Q column of FPLC system. Pooled hemolytic peak from CM-Sepharose-CL-6B was applied to the column and chromatographed as described in the text. The collected fractions were assayed for streptolysin 0 activity.

the supernatants were reprecipitated with higher concentrations of ammonium sulphate. The precipitates were dissolved in a small volume of 5 mmol/l phosphate buffer containing 0.5 mmol/l EDTA, and dialyzed overnight against the same buffer at 4°C. Table 1 shows a summary of the obtained results. Ammonium sulphate at 65% of saturation and pH 7.4 was selected for this concentration step. A concentration of the culture supernatant of nearly 30-fold was achieved in this precipitation. The dialyzate of the 65% ammonium sulphate saturation pH 7.4 fraction was then precipitated by the fractionated addition of solid polyethylene glycol 6000. The dialyzate was previously adjusted to different pH values as indicated above. After 60 min at room temperature the mixtures were centrifugated at 10,OOOgfor 20 min, and the supernatants were preof cipitated again with higher concentrations polyethylene glycol. The precipitates were redissolved in a small volume of 5 mmol/l phosphate buffer containing 0.5 mmol/l EDTA. Table 2 shows a summary of the results obtained. Protein did not precipitate at pH values lower than 8.0. Addition of polyethylene glycol to 20% saturation at pH 8.0 was selected. A further 7-fold concentration of the material was achieved with this second precipitation step. Pur$cation

4

6

mg protein/ml

8

gel

Fig. 2. Effect of protein applied to CM-Sepharose-CL-6B on the recovery and the purification degree.

of streptolysin 0

Streptolysin 0 was further purified from the culture supernatant concentrate by ion-exchange chromatography. Chromatography on DEAESephacel at pH 7.0 and 8.5 was first tested as described by other authors (Alouf and Raynaud, 1962, 1967, 1973; Bhakdi et al., 1984) but, in our tests, streptolysin 0 was only partially retained and the recoveries were consequently poor. However, cation-exchange chromatography on CM-Sepharose

1076

FRANCESCACANALIASet al.

Table 3. Summary of the purification procedure of streptolysin 0 Protein

Fraction Culture supernatant AS precipitation PEG precipitation CM-Seuharose-CL-6B Concenke AS Mono Q

(mg) 35800 1626 261 II 4

I

Act.

WU) 2200 2000 1176 1026 864 415

Yield Sp. act. (%) WU/mg) loo 91 81 47 39 19

0.06 I .23 6.80 93 216 415

Purification (fold)

I 20 II3 1554 3600 6917

AS: ammonium sulphate; PEG: polyethylene glycol.

at pH 5.5 completely retained the exoenzyme which could then be eluted with a salt gradient. Different amounts of concentrate were adjusted to pH 5.5 by dropwise addition of 1 mol/l acetic acid and applied to a CM-Sepharose-CL-6B column (5 x 60 cm) equilibrated with 5 mmol/l phosphate buffer pH 5.5 containing 0.5 mmol/l EDTA. The column was washed with 2 volumes of the same buffer and eluted with a linear salt gradient of NaCl from 0 to 0.6mol/l in the same phosphate buffer. Streptolysin 0 eluted in one single peak at a NaCl concentration of 0.25 mol/l (Fig. 1). Figure 2 shows the effect of the protein load on the recovery and extent of purification. From the results, it was decided to use 1 ml of packed gel per 10 mg of protein present in the sample. All chromatographic procedures were carried out at 4’C. Fractions containing hemolytic activity were pooled and brought to 65% saturation with solid ammonium sulphate, pH 7.4 at 4°C. The precipitate obtained by centrifugation at 10,000 g for 20 min was redissolved in a small volume of 20 mmol/l Tris/HCl buffer pH 8.0 and dialyzed overnight at 4C against the same buffer. The dialyzate was then chromatographed in a Mono Q (HR 5/5) column attached to an FPLC system. The 1 ml column was equilibrated with 20 mmol/l Tris/HCl buffer pH 8.0 and the sample was previously filtered through an 0.45pm Millex HV filter. Flow rate was 1 ml/min. The column was washed with the equilibration buffer, and streptolysin 0 was eluted with the same buffer using a discontinuous gradient of NaCl (Fig. 3). The fractions with hemolytic activity were pooled and stored frozen at -80°C in small aliquots. Table 3 shows the extent of purification and yields at different stages of the described purification procedure. Data shown are the mean of 15 different purifications. The described procedure for streptolysin 0 purification from culture supernatant of Streptococcus pyogenes appeared to be very reproducible in obtaining a final purified material with similar specific activity, even when the starting culture supernatants had different streptolysin 0 concentrations. Purity and physical characterization of the isolated toxin

Purified streptolysin mol. wt 60,100 and

0 showed a major band of several other minor bands

on polyacrylamide gel electrophoresis in the presence of SDS (Fig. 4). A trace amount of DNA-hydrolase activity was detected in the purified preparation. Molecular weight of streptolysin 0 was also determined by gel filtration chromatography in a Sephacryl S-200 SF calibrated with proteins of known molecular weight. Streptolysin 0 activity was found in one single peak which eluted at a volume corresponding to a molecular weight of 60,200. Isoelectric focusing of the purified material on polyacrylamide gels showed a major band at pH 7.5 with streptolysin 0 activity and several other minor bands, which were not hemolytically active. Effect of various factors on the hemolytic activity of the purified streptolysin 0 Erythrocyte concentration. A fixed amount of purified streptolysin 0 (1.8 HU/ml) was incubated at 30°C for 30min with different concentrations of rabbit erythrocytes, and the hemolytic activity was determined as described in Materials and Methods. Figure 5 shows the obtained results. The optimum erythrocyte concentration in the reaction mixture was 3.9 x 10’ cell/ml. Higher concentrations inhibited the streptolysin 0 hemolytic activity. pH. The effect of varying the pH on the purified streptolysin 0 activity assay was determined in 36 mmol/l phosphate buffer containing 126 mmol/l sodium chloride. Maximum hemolysis was obtained at pH 7.0 (Fig. 6). Incubation time and temperature. The hemolysis obtained was proportional to the incubation time for at least 200min under the conditions shown in Methods for the streptolysin 0 assay and when the hemolysis is lower than 50% (Fig. 7). The percentage of hemolysis increased with temperature in the range from 17 to 37°C (Table 4). Antitetanus toxin. The hemolytic activity of purified streptolysin 0 on erythrocytes was inhibited by the addition of antitetanus toxin in the reaction mixture. Fixed amounts of streptolysin 0 (2 HU/ml) and erythrocytes (0.39 x 10acell/ml) were incubated at 30°C with variable concentrations of antitetanus toxin (0.5-50 IU). Antitetanus toxin concentrations higher than 5.0 IU inhibited the hemolytic action of streptolysin 0 on the erythrocytes (Table 5). DISCUSSION

Streptolysin 0 is considered an important phatogenie factor of p-hemolytic group A streptococci.

Streptolysin 0 purification

1077

‘.__.

-w 1

2

3

4

5--

Fig. 4. Polyacrylamide gel electrophoresis in presence of SDS (protein concentration was 20 peg in each lane). Lane 1: molecular weight markers; lane 2: culture supernatant; lane 3: concentrate from the culture supematant; lane 4: CM-Sepharose-CL-6B; lane 5: Mono Q.

FRANCESCA

1078

CANALIAS

et

al.

100

20

C

I 0.5

I

I

I

I

I

10

15

20

2.5

30

Erythrocytes

Fig. 5. Effect of erythrocyte

I 35

0

concentration activity.

0

on streptolysin

I

I

I

I

65

70

7.5

80

PH Fig. 6. Effect of pH on streptoiysin

0 activity

tmin)

time on streptolysin

0 activity.

which is simpler and more reproducible than those previously described by other authors, and that can be scaled up easily. The large volume of the culture supernatant of Streptococcus pyogenes (which could be as large as 1000 1in industrial fermentations) is reduced to about l/l00 by serial precipitation with ammonium sulphate and polyethylene glycol. This is a critical step in the obtention of more operational volumes for the subsequent chromatographic procedures and has been carefully optimized to obtain maximum recovery as well as purification of streptolysin 0 out from other proteins and peptides of the culture medium. Chromatography of the concentrate on CMSepharose at pH 5.5 has been found to be more efficient and reproducible than chromatography on anionic gels as described by other authors (Alouf and Raynaud, 1962, 1973; Bhakdi rt al., 1984). The effect of protein load on the recovery and purification degree of streptolysin 0 has been carefully studied due to the impact of this factor on the overall cost of the chromatographic step. Although the obtained toxin is pure enough after the CM-chromatography to be used in the diagnostic reagents used for the determination of antistreptolysin 0 (ASLO), purification was improved by adding a FPLC step on a Mono Q resin. The degree of purification and yield finally obtained for streptolysin 0 compare favourably with those given by other authors (Table 6), although the preparation showed several contaminants in SDS-PAGE. The molecular weight and isoelectric

tained I

I 200

Fig. 7. Effect of incubation

Table

C’

60

I 150

time

L

55

I 100

per mL (x10’)

The purified toxin has interest in the manufacture of diagnostic reagents for the determination of antistreptolysin 0 (ASLO) in the sera of patients suspected to suffer acute glomerulonephritis and rheumatic fever caused by streptococcal infections. The purification of streptolysin 0 from culture su~rnatants of Streptococcus pyogenes has been described by several authors (Herbert, 1941; Pentz and Shigemura, 1955; Alouf and Raynaud, 1962, 1967, 1973; Van Epps and Andersen, 1969; Prigent et al., 1978; Linder, 1979; Gazzei et al., 1982; Bhakdi et al., 1984). Nevertheless, these procedures show poor reproducibility and are difficult to scale up for the production of large amounts of toxin. We have developed an optimized procedure for streptolysin 0 purification from Streptococcus pyogenes culture

>-

/ 50

4. Percentage of incubating

erythrocytcs

hemolysis

streptolysin

0

oband

for 20 min at different temDeratures

Tempereture ( C) Hemolysis 1%)

0 0

4 0

17 30 37 II 5x 85

1079

Streptolysin 0 purification Table 5. Effect of

antitetanus toxin on streptolysin 0 activity

Antitetanus toxin (IU) Hemolvsis (%)

0 90

0.5

5.0

89

50

IO

8

the enzyme and its separation from streptolysin 0. J. exp. Med. 106, 15-26. Cowell J. L. and Bernheimer A. W. (1977) Antigenic relationships among thiol-activated cytolysins. Infect. Immun. 16, 397-399.

Table 6. Comparison of streptolysin 0 purification results obtained by different authors Purification (fold)

Yield (%)

1939 3012 28 8 -

3 6 3 89 IO I9

Aloof and Raynaud (1962) Alouf and Raynaud (1973) Linder (1979) Gauei er al. (1982) Bhakdi ef al. (1984) This report

6917

637-638.

point of the purified toxin was found to be similar to those previously described (Alouf and Raynaud, 1973; Van Epps and Andersen, 1969; Bhakdi et al., 1984; Shany et al., 1973; Smyth and Fehrenbach, 1974; Suzuki et al., 1988). In the study of the effect of pH on the hemolytic activity of the purified streptolysin 0, we obtained a maximum hemolysis at pH 7.0. Herbert (1941) proposed a optimum pH of 6.5, although the difference in activity between these two pH is small (Fig. 7). The optimum erythrocyte concentration in the streptolysin 0 activity determination was 3.9 x 10’ cell/ml, a value in agreement with those given by Herbert (1941) and Kanbayashi et al. (1972). The hemolytic action of streptolysin 0 on the erythrocytes was found to be dependant of temperature showing an increase of IO-15% per “C.

661-717.

Herbert D. (1941) A simple calorimetric method for the estimation of haemolysis and its application to the study of streptolysin. Biochem. J. 35, 1116-I 123. Herbert D. and Todd E. W. (1941) Purification and properties of a haemolysin produced by group A haemolytic streptococci (streptolysin 0). Biochem. J. 35, 1124-l 139. Hewitt L. F. and Todd E. W. (1939) The effect of cholesterol and of sera contaminated with bacteria on the haemolysis produced by haemolytic streptococci. J. Path. Bucr. 49, 45-51. Kanbayashi Y., Hotta M. and Koyama J. (1972) Kinetic study on streptolysin 0. J. Biochem. 71, 227-237. Kaplan N. O., Colowick S. P. and Barnes C. C. (1951a) Effect of alkali on diphosphopyridine nucleotide. J. biol. Chem. 191, 461-472.

Kaplan N. 0.. Colowick S. P. and Nason A. (1951b) Neurospora diphosphopyridine nucleotidase. J. biol. Chem. 191, 473483. Klein G. C., Baker C. N., Addison B. V. and Moody M. D. (1969) Micro test for streptococcal anti-deoxyribonuclease B. Appl. Microbial. 18, 204206. Laemmli c K. (1970) Cleavage of structural proteins during the assembly of the head of Bacteriophage T4. Nature 227, 680685. Laemmli U. K. and Favre M. (1973) Maturation of the head of Bacteriophage T4. I. DNA packaging events. J. molec. Biol. 80, 575-599.

Linder R. (1979) Heterologous immunoaffinity chromatography in the purification of streptolysin 0. FEMS Microbiol. Lest. 5, 339-342.

REFERENCES

Alouf J. E. (1980) Streptococcal toxins.

Freifelder D. (1979) TPcnicos de Bioquimica y Biologia Molecular. Revert& Barcelona. Gazzei G., Palei F., Benanchi P. L., Fabbiani S., Antoni G. and Neri P. (1982) Large scale preparation of oxidized streptolysin 0 using molecular filtration. Experientiu 38,

Pharmac.

Ther. 11,

Alouf J. E. and Raynaud M. (1962) Purification of streptolysin 0. Nature 196, 374375. Alouf J. E. and Raynaud M. (1967) Nouvelle methode de purification de la streptolysin 0. C.r. Acad. Sci., Paris 264, 2524-2525.

Alouf J. E. and Raynaud M. (1973) Purification and some properties of streptolysin 0. Biochimie 55, 1187-I 193. Bernheimer A. W. (1974) Interactions between membranes and cytolytic bacterial toxins. Biochirn. biophys. Acta 344, 27-50.

Bernheimer A. W. (1976) Mechanisms in Bacterial Toxinology, pp. 85-97. Wiley, New York. Bhakdi S., Roth M., Sziegoleit A. and Tranum-Jensen J. (1984) Isolation and identification of two hemolytic forms of streptolysin 0. Infecl. Immun. 46, 394-400. Bradford M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254.

Carlson A. S., Kellner A., Bernheimer A. W. and Freeman E. B. (1957) A streptococcal enzyme that acts specifically upon diphosphopyridine nucleotide: characterization of

Nelson J., Ayoub E. M. and Wannamaker L. W. (1968) Streptococcal anti-desoxyribonuclease B: microtechnique determination. J. Lab. clin. Med. 71. 867-873. Pentz E. I. and Shigemura Y. (1955) The production, concentration and partial characterization of streptolysin 0. J. exp. Med. 69, 210-214. Prigent D., Geoffroy D. and Alouf J. E. (1978) Purification de la streptolysin 0 par chromatographie covalente sur gel de thiol-agarose. C.r. Acad. Sci., Paris 287, 951-954. Shany S., Grushoff P. S. and Bernheimer A. W. (1973) Physical separation of streptococcal nicotinamide adenine dinucleotide glycohydrolase from streptolysin 0. Infect. Immun. 7, 731-734.

Smyth C. J. and Fehrenbach F. J. (1974) Isoelectric analysis of haemolysins and enzymes from streptococci of groups A, C and G. Acta. path. microbial. stand., Sect. B. 82, 86Ck870.

Suzuki J., Kobayashi S., Kagaya K. and Fukazawa Y. (1988) Heterogeneity of hemolytic efficiency and isoelectric point of streptolysin 0. Infict. Immun. 5k, 2474-2478. Todd E. W. (1932) Antinenic strentococcal hemolvsin. J. exp. Med.‘55,

i67-286

-

Van Epps D. E. and Andersen B. R. (1969) Streptolysin 02 sedimentation coefficient and molecular weight determinations. J. Butt. 100, 526527.