Int. J. Biochem. Vol. 24,No. 7,pp.

1073-1079, 1992 0020-711X/92

$5.00+0.00
Printed in Great Britain. All rights reserved Copyright 0 1992Pcrgamon Press Ltd

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 streptococcal infection develop specific antibodies

Streptolysin 0 is a toxic immunogenic protein
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 oxi-
dation 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 sulphydryl-
activated cytolysins elaborated by gram-positive
bacteria. These toxins have several common proper-
ties (Bemheimer, 1974, 1976; Cowell and Bernheimer,
1977): they show maximal activity in the presence of
reducing agents containing thiol groups, their activi-
ties 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
to streptolysin 0 which can combine specifically
pro- 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, DNA-
hydrolases or proteases), the low amount of strep-
tolysin 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 of culture supernatant with ammonium sulphate at

different pH
Protein Act. Yield sp. act. Purification
Fraction (mg) (kHW (%) (kHU/mn) (fold)
Culture supernatant 30600 1364 100 0.044 I
pp 55% pH 6.5 590 420 31 0.712 I6
pp 80% pH 6.5 I754 564 41 0.321 7
sn 80% pH 6.5 2366 100 -

pp 40% pH 7.4 II7 31 2

pp 50% pH 7.4 314 580 42 1.85 42
pp 65% pH 7.4 1100 1275 93 1.16 26
pp 85% pH 7.4 1196 100
sn 85% pH 7.4 1875 -
pp 40% pH X.0 120 156 II 1.30 29
pp 60% pH 8.0 850 I857 136 2.18 49
pp 75% pH 8.0 x91 90 7
sn 75% DH 8.0 I857 60
Initial supernatant volume was I I (pp: precipitate; sn: supernatant)

MATERIALS AND METHODS
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 focus-
ing 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 sur-
rounded 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 con-
taining 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
suspension (1.58 x IO8cell/ml) in the same buffer and incu-
bated at 30°C for 20min. At the end of the incubation,
reaction mixtures were chilled with ice-water and cen-
trifuged. Hemoglobin concentration in the supematants was
determined spectrophotometrically at 541 nm. Blanks with-
out sample and controls of total hemolysis were also run.
The hemolysis of each sample was calculated by using the
following equation:
A sample - A blank
% Hemolysis = X 100
A total hemolysis - A blank
One hemolytic unit (HU) was defined as that amount of
streptolysin 0 which cause hemolysis of 50% of the erythro-
cytes (approx 3.95 x IO’) under the standard assay con-
ditions described. Hemolytic activity concentration was
calculated by interpolation of percentage of hemolysis in a
calibration plot.
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 sys-
tem 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 polyacryl-
amide 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

Table 2. Summary of the fractionation of culture supernatant with polyethylene glycol 6000
Protein Act. Yield Sp. act. Purification
Fraction (mg) (kHU) (%) (kHU/mg) (fold)
Culture supernatant 30600 I364 100 0.044 I
pp AS 65% pH 7.4 II00 1275 93 I.160 26
pp 20% pH 8.0 163 1027 75 6.300 143
pp 30% pH 8.0 134 5 - - -
sn 30% DH 8.0 588 - - -

Initial supernatant volume was I I (AS: ammonium sulphate; pp: precipitate; sn: supernatant).
 Streptolysin 0 purification 1075

03
0 Protein A260nm
0 Protem A260 “m
0 NoCl (mol/L)
0 NaCl imol/Ll
A SLO A541 nm

;
06 u 02
2
2

g 008 E
G
g
n
B 01
a
004
I

P
Fraction number
Froctlon number
Fig. I. Ion-exchange chromatography on CM-Sepharose-
CL-6B. Concentrate from the culture supernatant was ap- Fig. 3. Ion-exchange chromatography on Mono Q column
plied to the column and the chromatography was run as of FPLC system. Pooled hemolytic peak from CM-Sepha-
described in the text. The collected fractions were assayed rose-CL-6B was applied to the column and chromato-
for conductivity (NaCI mol/l), absorbance at 280 nm (pro- graphed as described in the text. The collected fractions were
tein) and absorbance at 541 nm (streptolysin 0 activity). assayed for streptolysin 0 activity.

after stirring overnight at 4‘C were assayed
activity.
RESULTS
Preparation of a concentrate from the culture super-
natant
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 super-
natant 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
for hemolytic the supernatants were reprecipitated with higher
concentrations of ammonium sulphate. The precipi-
tates 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 differ-
ent pH values as indicated above. After 60 min at
room temperature the mixtures were centrifugated at
10,OOOgfor 20 min, and the supernatants were pre-
cipitated again with higher concentrations of
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
40
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 of streptolysin 0
Streptolysin 0 was further purified from the
culture supernatant concentrate by ion-exchange
chromatography. Chromatography on DEAE-
Sephacel at pH 7.0 and 8.5 was first tested as
described by other authors (Alouf and Raynaud,
4 6 8 1962, 1967, 1973; Bhakdi et al., 1984) but, in our
mg protein/ml gel tests, streptolysin 0 was only partially retained and
Fig. 2. Effect of protein applied to CM-Sepharose-CL-6B the recoveries were consequently poor. However,
on the recovery and the purification degree. cation-exchange chromatography on CM-Sepharose
1076 FRANCESCACANALIASet al.
Table 3. Summary of the purification procedure of streptolysin 0
Protein
Fraction (mg)
Culture supernatant 35800
AS precipitation 1626
PEG precipitation 261
CM-Seuharose-CL-6B II
Concenke AS 4 864
Mono Q I 415
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 de-
cided 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 frac-
tions 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 pro-
cedure. Data shown are the mean of 15 different
purifications.
The described procedure for streptolysin 0 purifi-
cation from culture supernatant of Streptococcus
pyogenes appeared to be very reproducible in obtain-
ing 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 0 showed a major band of
mol. wt 60,100 and several other minor bands
Act. Yield Sp. act. Purification
WU) (%) WU/mg) (fold)
2200 loo 0.06 I
2000 91 I .23 20
1176 81 6.80 II3
1026 47 93 1554
39 216 3600
19 415 6917
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 deter-
mined 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 phato-
genie 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.
1078 FRANCESCA CANALIAS et al.

100

20

I I I I I I I / I I I
C 0.5 10 15 20 2.5 30 35 0 50 100 150 200

Erythrocytes per mL (x10’) time tmin)

Fig. 5. Effect of erythrocyte concentration on streptolysin 0 Fig. 7. Effect of incubation time on streptolysin 0 activity.
activity.

which is simpler and more reproducible than those

The purified toxin has interest in the manufacture of
diagnostic reagents for the determination of anti-
streptolysin 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
L those given by other authors (Table 6), although
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 sul-
phate 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 recov-
ery as well as purification of streptolysin 0 out from
other proteins and peptides of the culture medium.
Chromatography of the concentrate on CM-
Sepharose 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 antistrep-
tolysin 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
the preparation showed several contaminants in
SDS-PAGE. The molecular weight and isoelectric

Table 4. Percentage of hemolysis ob-

C’
tained incubating streptolysin 0 and
I I I I I erythrocytcs for 20 min at different tem-
>-
55 60 65 70 7.5 80 Deratures
PH Tempereture ( C) 0 4 17 30 37
Hemolysis 1%) 0 0 II 5x 85
Fig. 6. Effect of pH on streptoiysin 0 activity
 Streptolysin 0 purification 1079

Table 5. Effect of antitetanus toxin on streptolysin the enzyme and its separation from streptolysin 0. J. exp.
0 activity Med. 106, 15-26.
Antitetanus toxin (IU) 0 0.5 5.0 50 Cowell J. L. and Bernheimer A. W. (1977) Antigenic
Hemolvsis (%) 90 89 IO 8 relationships among thiol-activated cytolysins. Infect.
Immun. 16, 397-399.
Freifelder D. (1979) TPcnicos de Bioquimica y Biologia
Table 6. Comparison of streptolysin 0 purification Molecular. Revert& Barcelona.
results obtained by different authors
Gazzei G., Palei F., Benanchi P. L., Fabbiani S., Antoni G.
Purification Yield and Neri P. (1982) Large scale preparation of oxidized
(fold) (%) streptolysin 0 using molecular filtration. Experientiu 38,
Aloof and Raynaud (1962) 1939 3 637-638.
Alouf and Raynaud (1973) 3012 6 Herbert D. (1941) A simple calorimetric method for the
Linder (1979) 28 3 estimation of haemolysis and its application to the study
Gauei er al. (1982) 8 89 of streptolysin. Biochem. J. 35, 1116-I 123.
Bhakdi ef al. (1984) - IO
Herbert D. and Todd E. W. (1941) Purification and proper-
This report 6917 I9
ties 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
point of the purified toxin was found to be similar to and of sera contaminated with bacteria on the haemolysis
those previously described (Alouf and Raynaud, produced by haemolytic streptococci. J. Path. Bucr. 49,
1973; Van Epps and Andersen, 1969; Bhakdi et al., 45-51.
Kanbayashi Y., Hotta M. and Koyama J. (1972) Kinetic
1984; Shany et al., 1973; Smyth and Fehrenbach,
study on streptolysin 0. J. Biochem. 71, 227-237.
1974; Suzuki et al., 1988). Kaplan N. O., Colowick S. P. and Barnes C. C. (1951a)
In the study of the effect of pH on the hemolytic Effect of alkali on diphosphopyridine nucleotide. J. biol.
activity of the purified streptolysin 0, we obtained a Chem. 191, 461-472.
maximum hemolysis at pH 7.0. Herbert (1941) Kaplan N. 0.. Colowick S. P. and Nason A. (1951b)
Neurospora diphosphopyridine nucleotidase. J. biol.
proposed a optimum pH of 6.5, although the differ- Chem. 191, 473483.
ence in activity between these two pH is small (Fig. 7). Klein G. C., Baker C. N., Addison B. V. and Moody M. D.
The optimum erythrocyte concentration in the strep- (1969) Micro test for streptococcal anti-deoxyribonucle-
tolysin 0 activity determination was 3.9 x 10’ cell/ml, ase B. Appl. Microbial. 18, 204206.
Laemmli c K. (1970) Cleavage of structural proteins
a value in agreement with those given by Herbert during the assembly of the head of Bacteriophage T4.
(1941) and Kanbayashi et al. (1972). The hemolytic Nature 227, 680685.
action of streptolysin 0 on the erythrocytes was Laemmli U. K. and Favre M. (1973) Maturation of the head
found to be dependant of temperature showing an of Bacteriophage T4. I. DNA packaging events. J. molec.
Biol. 80, 575-599.
increase of IO-15% per “C.
Linder R. (1979) Heterologous immunoaffinity chromatog-
raphy in the purification of streptolysin 0. FEMS Micro-
REFERENCES biol. Lest. 5, 339-342.
Nelson J., Ayoub E. M. and Wannamaker L. W. (1968)
Alouf J. E. (1980) Streptococcal toxins. Pharmac. Ther. 11, Streptococcal anti-desoxyribonuclease B: microtechnique
661-717. determination. J. Lab. clin. Med. 71. 867-873.
Alouf J. E. and Raynaud M. (1962) Purification of strep- Pentz E. I. and Shigemura Y. (1955) The production,
tolysin 0. Nature 196, 374375. concentration and partial characterization of streptolysin
Alouf J. E. and Raynaud M. (1967) Nouvelle methode de 0. J. exp. Med. 69, 210-214.
purification de la streptolysin 0. C.r. Acad. Sci., Paris Prigent D., Geoffroy D. and Alouf J. E. (1978) Purification
264, 2524-2525. de la streptolysin 0 par chromatographie covalente sur
Alouf J. E. and Raynaud M. (1973) Purification and some gel de thiol-agarose. C.r. Acad. Sci., Paris 287, 951-954.
properties of streptolysin 0. Biochimie 55, 1187-I 193. Shany S., Grushoff P. S. and Bernheimer A. W. (1973)
Bernheimer A. W. (1974) Interactions between membranes Physical separation of streptococcal nicotinamide adenine
and cytolytic bacterial toxins. Biochirn. biophys. Acta 344, dinucleotide glycohydrolase from streptolysin 0. Infect.
27-50. Immun. 7, 731-734.
Bernheimer A. W. (1976) Mechanisms in Bacterial Toxinol- Smyth C. J. and Fehrenbach F. J. (1974) Isoelectric analysis
ogy, pp. 85-97. Wiley, New York. of haemolysins and enzymes from streptococci of groups
Bhakdi S., Roth M., Sziegoleit A. and Tranum-Jensen J. A, C and G. Acta. path. microbial. stand., Sect. B. 82,
(1984) Isolation and identification of two hemolytic forms 86Ck870.
of streptolysin 0. Infecl. Immun. 46, 394-400. Suzuki J., Kobayashi S., Kagaya K. and Fukazawa Y.
Bradford M. (1976) A rapid and sensitive method for the (1988) Heterogeneity of hemolytic efficiency and isoelec-
quantification of microgram quantities of protein utilizing tric point of streptolysin 0. Infict. Immun. 5k, 2474-2478.
the principle of protein-dye binding. Analyt. Biochem. 72, Todd E. W. (1932) Antinenic strentococcal hemolvsin.
248-254. J. exp. Med.‘55, i67-286 -
Carlson A. S., Kellner A., Bernheimer A. W. and Freeman Van Epps D. E. and Andersen B. R. (1969) Streptolysin 02
E. B. (1957) A streptococcal enzyme that acts specifically sedimentation coefficient and molecular weight determi-
upon diphosphopyridine nucleotide: characterization of nations. J. Butt. 100, 526527.