INFECTION AND IMMUNITY, Sept. 1976, p. 726-735 Copyright © 1976 American Society for Microbiology
Vol. 14, No. 3
Printed in U.S.A.
Purification and Properties of Streptococcal Hyaluronate Lyase JOHN HILL Department of Microbiology, The University of Chicago, Chicago, Illinois 60637 Received for publication 4 May 1976
Hyaluronate lyase (hyaluronidase)'has been purified and characterized from a group A type 4 Streptococcus. Production of the enzyme was favored by growth in trypsinized veal infusion in the presence of hyaluronate oligosaccharide and tetrasaccharide. Detectable enzymatic activity was diminished in the presence of N-acetylglucosamine and glucuronic acid. Purification of hyaluronate lyase consisted of 40 to 60% ammonium sulfate precipitation, diethylaminoethyl A-50 Sephadex ion-exchange chromatography, gel filtration with G-200 Sephadex, and adsorption to Sepharose 6B. Purified enzyme was antigenically homogeneous and free of proteinase, deoxyribonuclease, streptolysin 0, and streptokinase. Active hyaluronate lyase was recovered from neutral polyacrylamide gels, and it appeared to be a glycoprotein. A single band was detected by sodium dodecyl sulfate-acrylamide electrophoresis, which had a molecular weight of approximately 50,000. A molecular weight of 70,000 was observed by gel filtration. The purified enzyme had a Km of 3.8 x 10-4 and a pH optimum of 6.0. Reducing agents increased the activity of crude enzyme at least threefold and were necessary to prevent inactivation of the purified enzyme.
Hyaluronate lyase (EC 4.2.2.1) is produced by virtually all pathogenic streptococci belonging to groups A, B, C, and G (12, 21). Even though the function of the enzyme is uncertain, it is still of interest for several reasons. First, it is thought that the enzyme may play some role in the invasion of streptococci. Second, the elevation of anti-hyaluronidase antibody titer (AHT) in patients is one of the main indicators of a recent streptococcal infection. Third, the capsular material of streptococci is the substrate of this enzyme (20). Other bacteria also produce hyaluronate lyase, but none has been investigated as thoroughly in terms of virulence and invasiveness. Some strains of Staphylococcus aureus produce appreciable amounts of hyaluronate lyase. The characterization of the staphylococcal enzyme suggests that it is an isoenzyme, and Abramson and Friedman have recently reported that there may be a correlation between enzyme form and the type of infection from which it was isolated (1-4). It has been reported that the streptococcal enzyme is also an isoenzyme; however, this observation has not been confirmed with enzyme preparations of demonstrated purity. The streptococcal enzyme has not been purified beyond salt fractionation and ion-exchange chromatography (14, 22). It should be clear that streptococcal lyase cannot be characterized as
an isoenzyme after such a limited purification. The following is a description of the purification and the properties of hyaluronate lyase produced by a single Grifflth, type 4 Streptococcus. This strain was chosen because of its ability to produce high levels of enzyme under conditions where relatively low amounts of other extracellular productions were produced. MATERIALS AND METHODS Hyaluronate lyase determinations. The enzyme was assayed by the turbidimetric method of Kass and Seastone (16), modified by the use of heat-denatured 1.0% Metrix bovine albumin (Armour Pharmaceutical Co., Chicago, Ill.) at pH 3.0 in the place of horse serum. The enzyme was also determined by the spectrophotometric and colorimetric assays described by Greiling (13). The spectrophotometric assay was routinely used because of its superior sensitivity and speed. The protein concentration of enzyme preparations was determined spectrophotometrically as described by Layne (17). Hyaluronic acid. Solutions of purified streptococcal hyaluronic acid were obtained as a gift from Alvin Markovitz (Department of Microbiology, University of Chicago). Potassium hyaluronate was obtained from Gallard-Schlesinger (New York, N.Y.) for standardization by a carbazole test for uronic acids as described by Davidson (9). Enzyme production. A lyophilized culture of a beta-hemolytic Streptococcus pyogenes, Griffith type 4, was obtained from the American Type Culture
726
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STREPTOCOCCAL HYALURONATE LYASE
Collection (ATCC) as strain 10402. It was originally isolated from a throat infection and studied by Pike (24) because of its ability to produce large quantities of hyaluronate lyase in broth cultures. The basic growth medium was veal infusion broth (Difco Laboratories, Detroit, Mich.) which had been pretreated with Difco trypsin (1:250) for 3 h at 37°C. Media were supplemented with the degradation products and the constituents of hyaluronic acid. The oligosaccharide of hyaluronate was reduced to the tetrasaccharide form by the action of ovine testicular hyaluronidase (EC 3.2.1.35) and purified by the method of Ludowieg et al. (19). A disaccharide of hyaluronate was prepared by digesting hyaluronate with hyaluronate lyase (EC 4.2.2.1) until the further addition of enzyme to the substrate no longer resulted in an increase in degradation product as determined spectrophotometrically. The reaction mixture was dialyzed against deionized water, and the carbohydrate passing through the membrane was flash evaporated. Enzymatic digestion was repeated until a single spot corresponding to the disaccharide was detectable by thin-layer chromatography according to the technique of Linker et al. (18). Glucuronic acid and N-acetylglucosamine, the subunit sugars of hyaluronic acid, were obtained from Sigma (St. Louis, Mo.). Crude hyaluronate lyase intended for purification and characterization was prepared in no less than 15-liter batches. Strain 10402 was first subcultured seven times in media supplemented with 600 ,ug of hyaluronic acid per ml for enzyme induction. A 1:100 inoculum from an induced culture was used to inoculate trypsinized veal infusion without supplement. After 18 h of stationary incubation at 37°C, this culture was diluted 10-fold and incubation was continued for 6 h. Culture fluids containing extracellular products were separated from cells by continuous-flow centrifugation (Ivan Sorvall, Inc., Norwalk, Conn.). This supernatant was designated as fraction I enzyme. Enzyme purification. Fraction I enzyme was harvested from the culture fluids by ammonium sulfate fractional precipitation in the cold. The 40 to 60% fraction was collected with a Sharples Super centrifuge (Philadelphia, Pa.) and dissolved in the smallest possible volume of distilled water before extensive dialysis. This was fraction II enzyme. The enzyme was further purified by ion-exchange chromatography. Diethylaminoethyl (DEAE) A-50 Sephadex (Pharmacia Fine Chemicals, Piscataway, N.J.) was used in a column (1.5 by 33 cm). The column was eluted with the buffers described by Greiling (13); however, a continuous gradient was used instead of a step gradient to increase sodium chloride concentration. A batch technique was used for preparative purification of the enzyme. Enzyme preparations containing 12 mg of protein per ml were mixed with 2 volumes of gel equilibrated with 0.02 M phosphate buffer adjusted to pH 7.0. After the gel was washed with this buffer several times on a Buchner funnel, the enzyme was eluted from the gel by eight cycles of washing with 0.05 M NaH2PO4 (pH 6.0) containing 0.08 M NaCl. Enzyme was recovered from the washings by saturation with ammonium sulfate to 80%.
727
The precipitate was dissolved in the smallest possible volume of distilled water and dialyzed against 0.15 M sodium chloride containing 0.02% sodium azide as preservative and 1 mM dithiothreitol (Sigma) to stabilize the enzyme (fraction III). Fraction III enzyme was obtained from a total of 60 liters of fraction I and frozen at - 20°C. Pooled and concentrated fraction III enzyme was further purified by passage through an ascending column (5 by 88 cm) containing Sephadex G-200 (Pharmacia) equilibrated with the saline solution described above. Enzymatically active fractions were pooled and concentrated by dehydration with polyethylene glycol 20-M (Union Carbide, New York, N.Y.) and by ultrafiltration with collodion bags (Schleicher and Schuell, Keene, N.H.). This enzyme fraction was subsequently passed through a Sephadex G-150 column (2.5 by 37 cm), and a symmetric enzymatically active protein peak was observed. This was fraction IV enzyme. Fraction IV enzyme contained one major component and one or two minor components as determined by electrophoresis and gel diffusion techniques, depending on the enzyme batch. The remaining contaminants were removed by passing fraction IV enzyme through a Sepharose 6B (Pharmacia) column (1.5 by 33 cm) equilibrated with saline. Molecular weight determination. The molecular weight of fraction IV hyaluronidase was estimated on a Sephadex G-150 column (2.5 by 39 cm) which had been calibrated with aldolase, ovalbumin, chymotrypsinogen, and ribonuclease from a Pharmacia calibration kit. Elution values were plotted on a selectivity curve against the log of molecular weight according to the method of Andrews (5). Molecular weight was also estimated by sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis (8.5% slab gel) as described by Neville (23). The molecular weight was estimated by comparing the relative migration of purified hyaluronate lyase to the migration of bovine serum albumin (Armour), ovalbumin (Sigma), and lysozyme (Sigma). Relative migration was plotted against the log of molecular weight. Polyacrylamide gel electrophoresis. To demonstrate enzymatic activity after electrophoresis, polyacrylamide gels were run in an E-C vertical gel electrophoresis cell C 4 74 (St. Petersburg, Fla.). Both the 5% gel and the pH 7.0 buffer were prepared according to E-C technical bulletin no. 134. The electrophoresis buffer was modified by the addition of dithiothreitol (2 mM). After 3 h of pre-electrophoresis and a change of buffer, purified enzyme was electrophoresed at 120 mA for up to 9 h. The electrode buffer was constantly circulated, and the gel was maintained at 4 to 10°C to prevent enzyme inactivation. After electrophoresis, gels were stained for protein with 0.2% amido black 10B or were stained for carbohydrate according to the procedure of Fairbanks et al. (11). Duplicate gels were cut into 2.4-mm segments, mascerated in Sorensen's buffer (pH 6.0), and assayed for enzymatic activity after 12 h. Enzyme kinetics. The Michaelis constant was de-
728
HILL
INFECT. IMMUN.
termined for fraction IV enzyme with the tetrasaccharide of hyaluronate as substrate. The substrate concentration ranged from 0.074 to 2.4 mM tetrasaccharide in pH 6.0, 0.5 phosphate buffer. The reaction mixture was incubated for 1 h at 37°C, and the product was determined spectrophotometrically. The Km was determined from a Lineweaver-Burk reciprocal plot (10). Determination of other streptococcal products. Proteinase was determined by casein hydrolysis (7). Deoxyribonuclease (DNase) B (ADB) and streptolysin 0 (ASO) were determined with a commercial serological test kit (Beckman, Fullerton, Calif.). Nuclease activity was also determined by depolymerization of tritiated Escherichia coli deoxyribonucleic acid (DNA) obtained from N. Cozzarelli (Department of Biochemistry, University of Chicago). The labeled DNA had
a
specific activity of 2.3
x
108
,umol in pH 7.5, 30 ,iM tris(hydroxymethyl)aminomethane and 0.5 M ethylenediaminetetraacetic acid. The enzyme preparations were incubated for 2 h with the labeled DNA before chilling and the addition of carrier salmon testes DNA (Worthington). After thorough mixing, 2.5 M perchloric acid was added at 0°C and incubated for 10 min before removal of undigested DNA by centrifugation. A comparison was made between the nonprecipitable counts for the crude and purified enzyme preparations in toluene-based scintillation fluid. Streptokinase activity was determined with a (serological) kinase test kit from Fujizaki Pharmaceutical Co. (Tokyo, Japan). Hyaluronate lyase antisera. The fraction III enzyme was mixed with an equal volume of complete Freund adjuvant so that the final protein concentration was 0.06 mg/ml. The antigen was subcutaneously injected at two sites in 0.5-ml quantities into New Zealand White rabbits once a week over a 4week period before bleeding. Antihyaluronate lyase titer was determined with a Difco AHT kit and by a modification of the spectrophotometric assay for hyaluronic acid (13). In one row of tubes, 0.25 ml of immune serum was diluted from 1:32 to 1:2048. A duplicate row of tubes was made with serum from the corresponding rabbits before immunization. To each tube was added 0.25 ml of a 1:20 dilution of fraction III enzyme containing 1.2 mg of protein per ml. After 1 h of incubation at 37°C, 2.0 ml of a 0.2mg/ml, phosphate-buffered solution (pH 6.0) of hyaluronate was added to each tube and incubation was resumed for 10 min. The activity of free hyaluronate lyase was stopped by the addition of 0.5 ml of 20% perchloric acid at 0°C. The supernatant from each tube was decanted and the optical density was measured at 230 nm. AHT titer was recorded as the highest dilution of immune serum that had a lower optical density than normal serum. Immunodiffusion. The micro-Ouchterlony technique as described by Crowle (8) was used with 1.0% lonagar no. 2, buffered at pH 7.4 with 0.1 M phosphate. Human immune serum globulin was obtained from Hyland Laboratories (Costa Mesa, Calif.). Rabbit anti-hyaluronate lyase was prepared as described above. cpm per
RESULTS Production of hyaluronate lyase. More hyaluronate lyase was produced in veal infusion broth cultures than in Todd-Hewitt or brain heart infusion cultures. The dialyzable fraction of veal infusion broth as a medium yielded only 30% of the cells and 20% of the enzyme as the whole medium. Trypsinization of whole veal infusion broth resulted in a medium that yielded twice the hyaluronate lyase as the whole medium. In addition, trypsinization increased the efficiency of enzyme purification, especially at the gel filtration step. Cultures remained in log phase for 8 to 9 h. Peak enzyme production was during the 6th h and the enzyme level in cultures did not appreciably decline for 24 h. Agitation of cultures or the addition of 0.20% glucose increased cell densities, however, levels of detectable enzyme were reduced. Constant adjustment of the pH to 6.8 did not return hyaluronate lyase production to the normal level. The addition of 600 ug of hyaluronate or hyaluronate tetramer per ml increased total hyaluronate lyase activity by 40 and 20%, respectively (Table 1). Cultures containing these two substrates were uniformly less turbid at the end of log phase. The addition of hyaluronate dimer had a negligible effect on total enzymatic activity, whereas the addition of either N-acetylglucosamine or glucuronic acid reduced hyaluronate lyase activity by 40%. Enzyme purification. Ammonium sulfate precipitation and dialysis of the fraction I enTABLE 1. Hyaluronate lyase activation Relative OD600 of hyaluronConditioning substancea cultureb ate lyase yieldc 1.0 0.58 None 1.4 0.45 Hyaluronic acid 1.2 0.51 Tetramer 0.58 0.9 Dimer 0.6 0.58 N-acetylglucosamine 0.6 0.55 Glucuronic acid a Exponentially growing cells of strain 10402 were inoculated (1:100 dilution) into media containing one of each of the substrates. The substrates were present at 600 Ag/ml and were obtained as described in Materials and Methods. b Cultures were incubated to the end of log phase before cells were removed by centrifugation. OD, Optical density. c Relative amounts of hyaluronate lyase were determined spectrophotometrically in culture supernatants and after fractional ammonium sulfate precipitation.
STREPTOCOCCAL HYALURONATE LYASE
VOL. 14, 1976
729
TABLE 2. Purification of streptococcal hyaluronate lyase Fraction
Treatment
Total teinapro-
Enzyme Sp act uniac
I II III IV
None 0.32 kg 1.2 9.5 x 10-2 40-60% (NH4)2SO4 1.6 g 31.4 5.8 DEAE A-50 Sephadex 190 mg 317 48.8 Sephadex G-200 3.3 mg 323 248.5 a Protein determined according to Layne (17). b Hyaluronate lyase units were determined according to Greiling (13).
Punfication uifctin 0 1.2 x 102 9.1 X 102 1.2 x 104
Yield (% Yel
100 61 30 12
TABLE 3. Distribution of extracellular products in hyaluronate lyase preparations Prepne Product
Detection methoda
Crude
Hyaluronate lyase Streptokinase Streptolysin 0 DNase B
Pure
Serological
1:128 1:5,200 None Less than 40 1:40 None 1:160 Less than 1:40 DNA degradation 1 pmol/h per ,mI of DNA Negligible degradation Proteinase Casein digestion None None a Assay techniques are described in Materials and Methods. b Dilutions represent titration of hyaluronate lyase preparations against standard sera against other streptococcal products.
zyme reduced total protein to less than 1.0% of that in the crude broth. Seventy percent of the enzymatically active protein precipitated at 40 to 60% saturation and another 18% precipitated in the 60 to 80% saturation fraction. After each purification step, less enzyme was recovered by fractional precipitation (Table 2). When the protein concentration was below 200 ,ug/ml, ammonium sulfate precipitation could not be used for quantitative recovery of enzyme. Fraction III enzyme was obtained by ion-exchange chromatography of the dialyzed ammonium sulfate concentrates. On a DEAE'A-50 Sephadex column, about 80% of the enzymatically active protein eluted with 0.05 M phosphate at pH 6.0 (Fig. 1). The remaining enzyme eluted from the column with a sodium chloride gradient. Hyaluronate lyase eluting from the column before the salt gradient did not correspond to a protein peak, whereas the elution of enzyme with the NaCl salt gradient corresponded to the elution profile of protein. When the two hyaluronate lyase fractions were separately rechromatographed on DEAE A-50, they could not be distinguished. After ion-exchange chromatography, hyaluronate lyase was further purified on gel filtration columns. Hyaluronate lyase eluted from a preparative G-200 column as a single broad enzymatic peak; however, a distinct protein peak was not observed. Contaminants eluted after the enzyme as a single major peak. Enzymatically active fractions which were
pooled eluted as a single distinct protein peak when rechromatographed on a Sephadex G-150 column (Fig. 2). To obtain enzyme free of any detectable contaminants, fraction IV hyaluronate lyase was passed through a Sepharose 6B column. The enzyme was retarded on the column until contaminants had eluted. Determination of other streptococcal products. Fraction I enzyme contained appreciable amounts of DNase B but small amounts of streptokinase, streptolysin 0, and proteinase. Fraction IV enzyme was contaminated with only traces of DNase B, which were subsequently removed by Sepharose 6B purification. Gel diffusion on microscope slides revealed that crude hyaluronate lyase contained at least three antigens that were detectable with pooled human immune immunoglobulin G (Fig. 3). Specific rabbit antihyaluronate lyase also revealed more than one antigen, but the precipitin lines were indistinct. In contrast, neither antisera detected more than one antigen in purified enzyme preparations. AHTs of the specific rabbit antisera were determined with a commercial antihyaluronidase kit and by a spectrophotometric assay developed in this laboratory. Results of the enzyme blocking spectrophotometric assay were comparable to the commercial assay. One rabbit developed an AHT of 1,024 units, and two other animals developed antibody titers of 256 units. When the standard serum control in the commercial AHT kit was used, crude hyaluro-
730
INFECT. IMMUN.
HILL l150
~.140 130 -120 z
Io
-110 X
Go 0
s -80LU.
-ioo -
0 (.4
-90 0
0
- 70 60
z tu
I.Of 0
('
-
-50 it) 40 Z -30 0 ° 20 a
CL
10
FRACTIONS FIG. 1. DEAE-Sephadex A-50 chromatography of fraction II hyaluronate lyase. The first 40 fractions were eluted with 0.05 M phosphate (pH 6.0). Later fractions were eluted with a continuous salt gradient. Symbols: (0) Elution profile of proteins; (a) elution of enzymatic activity. The details of ion exchange are given in Materials and Methods. 130
.i12
.120
-~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ 910 Z'
.10
807
0.08
-~~~~~~~~~~~~~~~~70
OD
° Q .06
4
III
.
0
2
0
-60 u-, C.,
>
< ~~~~~~~~~~~50
-
LU U
14
16
18
20
22
24
26
28
30
30
0 cx.02 14
16
Z
-~~~~~~~~~~~~~~~~20 0 -10
18
20
22
26
24
28
30
32
c')
e4
0
FRACTIONS
FIG. 2. Elution profile of fraction IV hyaluronate
lyase
Symbols: (0) Protein elution profile; (a) enzymatic activity.
nate lyase had to be diluted 1:128 before hyaluronate-protein complexes would form. The purified enzyme had to be diluted 1:5,200 (Table 3). Molecular weight determination. A calibrated Sephadex G-150 column was used to estimate the molecular weight of hyaluronate lyase. The enzyme had a Ka. that corresponded to a molecular weight of 70,000 on a selectivity curve (Fig. 4). SDS-polyacrylamide gel elec'tro-
on a
Sephadex G-150 column (2.5 by 33 cm).
phoresis was also used to estimate molecular weight. A single band was detected on an 8.5% slab gel which had a relative migration (Rf) corresponding to a molecular weight of about 50,000 on a calibration curve of known markers (Fig. 5). Polyacrylamide gel electrophoresis. Active hyaluronate lyase could be recovered from polyacrylamide gels only under the conditions de-
VOL. 14, 1976
STREPTOCOCCAL HYALURONATE LYASE
scribed in the Materials and Methods. The use of basic buffers, temperatures above 14°C, and no circulation of buffer between electrode chambers resulted in enzyme inactivation. It was also necessary for gels to be prerun and reduced. Under conditions that permitted the recovery of active enzyme, gels could be stained for both protein and carbohydrate at positions corresponding to enzymatic activity (Fig. 6). Kinetics. The Michaelis constant for fraction IV enzyme with hyaluronate tetramer as sub-
731
strate was 3.8 x 10-4 M (Fig. 7). The tetrasaccharide was prepared by the enzymatic digestion of potassium hyaluronate of an unknown molecular weight with hyaluronidase from sheep testes. Further digestion of the tetrasaccharide with streptococcal hyaluronate lyase results in equimolar amounts of N-acetyl-hyalobiouronic acid and an unsaturated derivative of this compound which characteristically was produced by hyaluronate lyases. Optimal pH and enzyme stability. The opti-
FIG. 3. Micro-Ouchterlony study of purified (well b) and crude (well e) hyaluronate lyase with specific rabbit antiserum (wells a and d) and human immune immunoglobulin G.
732
HILL
INFECT. IMMUN.
potentiated fraction I enzyme by three-fold, and reducing agents were also necessary to stabilize fraction IV enzyme.
500
400 300
2001 a 0 100
x
3:
90 80 70 60
50
40
30
201
se
I I
I
I |
I I
I
.1
.3
.4
.5
.6
.2
.7
.8
.9
DISCUSSION A strain of group A, type 4, S. pyogenes was grown in the presence of several forms of hyaluronic acid and its constituents to determine whether hyaluronate lyase activity would be affected. These results were consistent with the reports that streptococcal hyaluronate lyase is an inducible enzyme; however, it has not been ruled out that activity rather than enzyme production was influenced (13, 22). Data also suggested that one hydrolyzable substrate bond must be present for there to be enzyme stimulation. It was observed that hyaluronate tetrasaccharide stimulated enzyme activity but the dimer did not, even though streptococci reportedly metabolize the dimer (18). N-acetylglucosamine and glucuronic acid, the constituents of hyaluronic acid, depressed hyaluronate activity by 40%, suggesting that hyaluronate lyase is subject to repression as well as activation. Sallet al. (25) reported that streptococci meIJgman tabolize these monosaccharides more vigor1.0
Kcv
1001
FIG. 4. Selectivity curve for the estimation of molecular weight by the method of Andrews (5) with a Sephadex G-150 column. Points a through e represent aldolyase, hyaluronate lyase, ovalbumin, chymotrypsinogen, and ribonuclease, respectively.
so 70
a
60
mal pH for the purified enzyme was 6.0. Fraction I enzyme did not drop drastically in percentage of activity until pH 5.0; however, fraction IV enzyme became less active immediately below its optimum pH and was irreversibly inactivated in 30 min at pH 5.5. On the basic side of the optimal pH, the percentage of activity declined more gradually. At pH 6.8, fraction IV enzyme was only 40% as active as at the optimal pH, but the enzyme remained stable so that full activity was observed when the pH was adjusted to the optimum. At pH values above 7.0, both the crude and purified enzyme became unstable, and irreversible loss of activity was observed within 24 h. At pH 8.6 all enzymatic activity was irreversibly lost after 10
501
b
NO 40 x
3¢ 30 20~
d
min.
After cells were removed from a culture, frac.3 .4 .7 .6 .8 .2 tion I enzyme retained full activity for 1 week, RF at room temperature if reducing agents were FIG. 5. SDS-acrylamide gel electrophoretic estipresent. Fraction IV enzyme lost 50% of its mation of molecular weight. Points a, b, c, and d activity after 3 h at room temperature. Both the represent the respective migrations of bovine serum purified and crude enzyme retained full activity albumin, hyaluronate lyase, ovalbumin, and lysofor 2 years at -20°C. Addition of dithiothreitol zyme on an 8.5% slab gel (pH 8.5). .5
STREPTOCOCCAL HYALURONATE LYASE
VOL. 14, 1976
, ,
, , ,,1-,,, ., , ,= "IC E
z
0
4n
' 15 20
10
5
' - .~ez 30 35 40 4S
25
MIGRATIONimm,
733
of this preparation in any way. Gel diffusion of fraction II enzyme revealed at least three antigens and SDS-gel electrophoresis revealed that up to 13 proteins were present. Although streptokinase and proteinase activities were eliminated by fractional ammonium sulfate precipitation, the resulting preparations were not pure. Fraction III enzyme is similar in preparation to the hyaluronate lyase described by Greiling et al. (13, 14). In agreement with these authors, it was observed that most of the enzyme eluted from ion-exchange columns with 0.05 NaH2PO4, but they also reported that a major protein peak contained the enzyme. In the current work, enzymatic activity was associated with a small fraction of the total protein (Fig. 1). A secondary enzymatic peak was associated with protein that eluted with a subsequent salt gradient. Greiling et al. (13, 14) reported heterogeneity in the elution of hyaluronate lyase, and they attributed this to multiple forms of the enzyme. Because the secondary enzymatic peak
6. Acrylamide gel electrophoresis of purified hyaluro. nate lyase on a 5% slab gel (pH 7.0). The top eluted with 0.05 NaH2PO4 when rechromatoof the fA gure (I) is enzymatic activity recovered from s. The bottom (II) is a densitometric scan of graphed, it seems most likely that there is a gel ) and carbohy- single form of the enzyme and the elution propduplicatte gels stained for protein ( -). (erties of it can be influenced by other proteins. * After purification by gel filtration, hyaluronate lyase eluted from Sephadex G-150 as a single enzymatically active protein peak, in contrast with staphylococcal hyaluronate lyase which eluted from Sephadex G-100 as multiple forms (2). At low ionic strengths the staphylococcal enzyme did not elute from Sephadex columns. This was not true of the streptococcal enzyme, where it was observed that elution was retarded on Sepharose 6B independently of Km 3.8 X 10-4 ionic strength. Sepharose 6B retention was utilized to remove contaminants from fraction IV , ,I i . , , ,I | , , , , _, slice
drate
22
20
18
16
14
12
10
0
2
4
6
8
10
12
14
16
I X io-3
s FIG. 7. Lineweaver-Burk plot for the determination oft 'he Michaelis constant ofpurified hyaluronate lyase w ith hyaluronate tetrasaccharide as substrate.
enzyme.
After purification of hyaluronate lyase by gel filtration, single lines of precipitation were formed with antisera in gel diffusion studies. In agreement with Halbert and Auerbach (15), human immune serum gamma globulin was
more effective in the detection of precipitating streptococcal antigens than specific antisera prepared in rabbits. In one case, specific rabbit oxidatiion influence the activity of hyaluronate antiserum detected one antigen in fraction II enzyme, whereas human gamma globulin lyase vvithout affecting its synthesis. Fraction II enzyme is comparable to the hy- formed four lines of precipitation against the aluron;ate lyase purified by Mogilevskii (22). same antigen. Polyacrylamide gel electrophoresis revealed After ammonium sulfate fractionation, his prepar;ations were treated with cholesterol to that a single protein species was present in purified enzyme preparations. Carbohydrate removEestreptolysin 0. Fraction II preparations contairied a small amount of streptolysin 0 and staining suggested that streptococcal hyalurolarge a mounts of DNase. Treatment with cho- nate lyase is a glycoprotein. Although no other lesterol1 did not alter the antigenic composition bacterial hyaluronate lyase has been character-
ously t;han hyaluronate oligosaccharide. It has not be en ruled out that the products of this
734
HILL
INFECT. IMMUN.
ized to this degree, it has been shown that testicular hyaluronidase is a glycoprotein with a molecular weight of 61,000 by gel filtration methods (6). The molecular weight of the streptococcal hyaluronate lyase was about 70,000 by gel filtration and 50,000 by SDS-acrylamide electrophoresis. No other molecular weight estimates for hyaluronate lyase are available for comparison; however, molecular weight determinations for testicular hyaluronidase range from 43,000 to 126,000, depending on enzyme purity and the method of determination (6). The Michaelis constant for fraction IV enzyme was 3.8 x 10-4 M when measured with a homogeneous substrate of a known molecular weight. Greiling et al. (14) reported Km values of 3.2 x 10-2 and 1.3 x 10-2 for streptococcal hyaluronate lyase resolved by ion-exchange chromatography. The difference between his two Km values is not great enough to indicate that two different enzymes having two different Km values were studied. Differences in purity alone would cause variations in Km of this magnitude because of different efficiencies of substrate binding. The difference between the Km reported here and those of Greiling is probably due to his use of an undefined oligosaccharide as substrate. The earlier values were obtained with the use of hyaluronic acid of an undesignated molecular weight, whereas a homogeneous substrate of a known molecular weight was used in this study. Abramson and Friedman (1-3) resolved three to four forms of staphylococcal hyaluronate lyase by chromatographic and electrophoretic techniques. This quality of staphylococcal hyaluronate lyase became significant to the study of infectious disease with the contention that a correlation between enzyme form and the clinical manifestations of staphylococcal disease could be observed. Greiling et al. (14) reported three hyaluronate lyases from staphylococci and two from streptococci. The results reported here indicate that ATCC strain 10402, a throat isolate, produced a single hyaluronate lyase as determined by chromatographic, electrophoretic, and serological techniques. This does not rule out the possibility that other streptococci produce multiple hyaluronate lyases. ACKNOWLEDGMENTS I wish to express my appreciation to William Martin for guidance and the use of his laboratory facilities. I also wish to thank Eugene Fox for his critical evaluation of this manuscript and V. Ormiste, R. Lewert, and M. Yogore for technical assistance. This investigation was supported by United States Public Health Service grant GM603 from the National Institute of General Medical Sciences.
LITERATURE CITED 1. Abramson, C. 1967. Staphylococcal hyaluronate lyase: multiple electrophoretic and chromatographic forms. Arch. Biochem. Biophys. 121:103-106. 2. Abramson, C. 1974. Staphylococcal hyaluronidase isoenzyme profiles related to staphylococcal disease. Ann. N.Y. Acad. Sci. 236:495-507. 3. Abramson, C., and H. Friedman. 1968. Staphylococcal hyaluronate lyase: purification and characterization studies. J. Bacteriol. 96:886-892. 4. Abramson, C., and H. Friedman. 1969. Electrophoretic characteristics of staphylococcal hyaluronate lyase. J. Bacteriol. 97:715-718. 5. Andrews, P. 1964. Estimation of molecular weights of proteins by Sephadex gel filtration. Biochem. J. 91:222-233. 6. Borders, C. P., and M. A. Raftery. 1968. Purification and partial characterization of testicular hyaluronidase. J. Biol. Chem. 243:3756-3762. 7. Cohen, J. 0. 1969. Effect of culture medium components and pH on the production of M protein and proteinase by group A streptococci. J. Bacteriol. 99:737-744. 8. Crowle, A. J. 1958. A simplified micro-double diffusion agar precipitin technique. J. Lab. Clin. Med. 52:784787. 9. Davidson, E. A. 1966. Analysis of sugars found in mucopolysaccharides, p. 54. In S. P. Colowich and N. 0. Kaplan (ed.), Methods in enzymology, vol. 8. Academic Press Inc., New York. 10. Dawes, E. A. 1969. Quantitative problems in biochemistry, 4th ed. The Williams and Wilkins Co., Baltimore. 11. Fairbanks, G., T. L. Steck, and F. H. Wallach. 1971. Electrophoretic analysis of the main polypeptides of the human erythrocyte membrane. Biochemistry 10:2606-2617. 12. Ginsberg, I. 1972. Mechanism of cell and tissue injury induced by group A streptococci: relation to poststreptococcal sequele. J. Infect. Dis. 126:294-340. 13. Greiling, H. 1963. Hyaluronic acid, p. 87-90. In H. Bergmeyer (ed.), Methods of enzymatic analysis. Academic Press, Inc., New York. 14. Greiling, H., H. W. Stuhlsatz, and T. Eberhard. 1965. Zur heterogenitat der hyaluronatlyase. Z. Physiol. Chem. 340:243-248. 15. Halbert, S. P., and T. Auerbach. 1961. The use of precipitin analysis in agar for the study of human streptococcal infections. IV. Further observations on the purification of group A extracellular antigens. J. Exp. Med. 113:131-157. 16. Kass, E. H., and C. V. Seastone. 1944. The role of the mucoid polysaccharide (hyaluronic acid) in the virulence of group A hemolytic streptococci. J. Exp. Med. 79:319-329. 17. Layne, E. 1957. Spectrophotometric and turbidimetric methods for measuring proteins, p. 451-454. In S. P. Colowich and N. 0. Kaplan (ed.), Methods in enzymology, vol. 3. Academic Press Inc., New York. 18. Linker, A., K. Meyer, and P. Hoffman. 1956. The production of unsaturated uronides by bacterial hyaluronidases. J. Biol. Chem. 219:13-25. 19. Ludowieg, J., B. Vennesland, and A. Dorfman. 1961. The mechanism of action of hyaluronidases. J. Biol. Chem. 236:333-339. 20. McCarty, M. 1973. Streptococci, p. 708-726. In B. D. Davis, R. Dulbecco, H. N. Eisen, H. S. Ginsberg, W. B. Wood, and M. McCarty (ed.), Microbiology, 2nd ed. Harper and Row, Hagerstown, Md. 21. Meyer, K., and M. M. Rapport. 1952. Hyaluronidases, p. 199-236. In F. F. Nord (ed.), Advances in enzymology, vol. 8. Interscience Pub., New York.
VOL. 14, 1976
STREPTOCOCCAL HYALURONATE LYASE
22. Mogilevskii, M. Sh. 1964. Isolation and purification of streptococcal hyaluronidase. Fed. PIfoc. 23:T559-561. 23. Neville, D. M. 1971. Molecular weight determination of protein-dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system. J. Biol. Chem. 246:6328-6334. 24. Pike, R. M. 1948. Streptococcal hyaluronic acid and
735
hyaluronidase. I. Hyaluronidase activity of noncapsulated group A streptococci. J. Infect. Dis. 83:1-11. 25. Sallman, B., J. M. Birkeland, and C. T. Gray. 1951. Hyaluronic acid utilized by hemolytic streptococci in relation to possible hyaluronidase function in pathogenesis. Proc. Soc. Exp. Biol. Med. 76:467-471.
Vol. 14, No. 3
Printed in U.S.A.
Purification and Properties of Streptococcal Hyaluronate Lyase JOHN HILL Department of Microbiology, The University of Chicago, Chicago, Illinois 60637 Received for publication 4 May 1976
Hyaluronate lyase (hyaluronidase)'has been purified and characterized from a group A type 4 Streptococcus. Production of the enzyme was favored by growth in trypsinized veal infusion in the presence of hyaluronate oligosaccharide and tetrasaccharide. Detectable enzymatic activity was diminished in the presence of N-acetylglucosamine and glucuronic acid. Purification of hyaluronate lyase consisted of 40 to 60% ammonium sulfate precipitation, diethylaminoethyl A-50 Sephadex ion-exchange chromatography, gel filtration with G-200 Sephadex, and adsorption to Sepharose 6B. Purified enzyme was antigenically homogeneous and free of proteinase, deoxyribonuclease, streptolysin 0, and streptokinase. Active hyaluronate lyase was recovered from neutral polyacrylamide gels, and it appeared to be a glycoprotein. A single band was detected by sodium dodecyl sulfate-acrylamide electrophoresis, which had a molecular weight of approximately 50,000. A molecular weight of 70,000 was observed by gel filtration. The purified enzyme had a Km of 3.8 x 10-4 and a pH optimum of 6.0. Reducing agents increased the activity of crude enzyme at least threefold and were necessary to prevent inactivation of the purified enzyme.
Hyaluronate lyase (EC 4.2.2.1) is produced by virtually all pathogenic streptococci belonging to groups A, B, C, and G (12, 21). Even though the function of the enzyme is uncertain, it is still of interest for several reasons. First, it is thought that the enzyme may play some role in the invasion of streptococci. Second, the elevation of anti-hyaluronidase antibody titer (AHT) in patients is one of the main indicators of a recent streptococcal infection. Third, the capsular material of streptococci is the substrate of this enzyme (20). Other bacteria also produce hyaluronate lyase, but none has been investigated as thoroughly in terms of virulence and invasiveness. Some strains of Staphylococcus aureus produce appreciable amounts of hyaluronate lyase. The characterization of the staphylococcal enzyme suggests that it is an isoenzyme, and Abramson and Friedman have recently reported that there may be a correlation between enzyme form and the type of infection from which it was isolated (1-4). It has been reported that the streptococcal enzyme is also an isoenzyme; however, this observation has not been confirmed with enzyme preparations of demonstrated purity. The streptococcal enzyme has not been purified beyond salt fractionation and ion-exchange chromatography (14, 22). It should be clear that streptococcal lyase cannot be characterized as
an isoenzyme after such a limited purification. The following is a description of the purification and the properties of hyaluronate lyase produced by a single Grifflth, type 4 Streptococcus. This strain was chosen because of its ability to produce high levels of enzyme under conditions where relatively low amounts of other extracellular productions were produced. MATERIALS AND METHODS Hyaluronate lyase determinations. The enzyme was assayed by the turbidimetric method of Kass and Seastone (16), modified by the use of heat-denatured 1.0% Metrix bovine albumin (Armour Pharmaceutical Co., Chicago, Ill.) at pH 3.0 in the place of horse serum. The enzyme was also determined by the spectrophotometric and colorimetric assays described by Greiling (13). The spectrophotometric assay was routinely used because of its superior sensitivity and speed. The protein concentration of enzyme preparations was determined spectrophotometrically as described by Layne (17). Hyaluronic acid. Solutions of purified streptococcal hyaluronic acid were obtained as a gift from Alvin Markovitz (Department of Microbiology, University of Chicago). Potassium hyaluronate was obtained from Gallard-Schlesinger (New York, N.Y.) for standardization by a carbazole test for uronic acids as described by Davidson (9). Enzyme production. A lyophilized culture of a beta-hemolytic Streptococcus pyogenes, Griffith type 4, was obtained from the American Type Culture
726
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STREPTOCOCCAL HYALURONATE LYASE
Collection (ATCC) as strain 10402. It was originally isolated from a throat infection and studied by Pike (24) because of its ability to produce large quantities of hyaluronate lyase in broth cultures. The basic growth medium was veal infusion broth (Difco Laboratories, Detroit, Mich.) which had been pretreated with Difco trypsin (1:250) for 3 h at 37°C. Media were supplemented with the degradation products and the constituents of hyaluronic acid. The oligosaccharide of hyaluronate was reduced to the tetrasaccharide form by the action of ovine testicular hyaluronidase (EC 3.2.1.35) and purified by the method of Ludowieg et al. (19). A disaccharide of hyaluronate was prepared by digesting hyaluronate with hyaluronate lyase (EC 4.2.2.1) until the further addition of enzyme to the substrate no longer resulted in an increase in degradation product as determined spectrophotometrically. The reaction mixture was dialyzed against deionized water, and the carbohydrate passing through the membrane was flash evaporated. Enzymatic digestion was repeated until a single spot corresponding to the disaccharide was detectable by thin-layer chromatography according to the technique of Linker et al. (18). Glucuronic acid and N-acetylglucosamine, the subunit sugars of hyaluronic acid, were obtained from Sigma (St. Louis, Mo.). Crude hyaluronate lyase intended for purification and characterization was prepared in no less than 15-liter batches. Strain 10402 was first subcultured seven times in media supplemented with 600 ,ug of hyaluronic acid per ml for enzyme induction. A 1:100 inoculum from an induced culture was used to inoculate trypsinized veal infusion without supplement. After 18 h of stationary incubation at 37°C, this culture was diluted 10-fold and incubation was continued for 6 h. Culture fluids containing extracellular products were separated from cells by continuous-flow centrifugation (Ivan Sorvall, Inc., Norwalk, Conn.). This supernatant was designated as fraction I enzyme. Enzyme purification. Fraction I enzyme was harvested from the culture fluids by ammonium sulfate fractional precipitation in the cold. The 40 to 60% fraction was collected with a Sharples Super centrifuge (Philadelphia, Pa.) and dissolved in the smallest possible volume of distilled water before extensive dialysis. This was fraction II enzyme. The enzyme was further purified by ion-exchange chromatography. Diethylaminoethyl (DEAE) A-50 Sephadex (Pharmacia Fine Chemicals, Piscataway, N.J.) was used in a column (1.5 by 33 cm). The column was eluted with the buffers described by Greiling (13); however, a continuous gradient was used instead of a step gradient to increase sodium chloride concentration. A batch technique was used for preparative purification of the enzyme. Enzyme preparations containing 12 mg of protein per ml were mixed with 2 volumes of gel equilibrated with 0.02 M phosphate buffer adjusted to pH 7.0. After the gel was washed with this buffer several times on a Buchner funnel, the enzyme was eluted from the gel by eight cycles of washing with 0.05 M NaH2PO4 (pH 6.0) containing 0.08 M NaCl. Enzyme was recovered from the washings by saturation with ammonium sulfate to 80%.
727
The precipitate was dissolved in the smallest possible volume of distilled water and dialyzed against 0.15 M sodium chloride containing 0.02% sodium azide as preservative and 1 mM dithiothreitol (Sigma) to stabilize the enzyme (fraction III). Fraction III enzyme was obtained from a total of 60 liters of fraction I and frozen at - 20°C. Pooled and concentrated fraction III enzyme was further purified by passage through an ascending column (5 by 88 cm) containing Sephadex G-200 (Pharmacia) equilibrated with the saline solution described above. Enzymatically active fractions were pooled and concentrated by dehydration with polyethylene glycol 20-M (Union Carbide, New York, N.Y.) and by ultrafiltration with collodion bags (Schleicher and Schuell, Keene, N.H.). This enzyme fraction was subsequently passed through a Sephadex G-150 column (2.5 by 37 cm), and a symmetric enzymatically active protein peak was observed. This was fraction IV enzyme. Fraction IV enzyme contained one major component and one or two minor components as determined by electrophoresis and gel diffusion techniques, depending on the enzyme batch. The remaining contaminants were removed by passing fraction IV enzyme through a Sepharose 6B (Pharmacia) column (1.5 by 33 cm) equilibrated with saline. Molecular weight determination. The molecular weight of fraction IV hyaluronidase was estimated on a Sephadex G-150 column (2.5 by 39 cm) which had been calibrated with aldolase, ovalbumin, chymotrypsinogen, and ribonuclease from a Pharmacia calibration kit. Elution values were plotted on a selectivity curve against the log of molecular weight according to the method of Andrews (5). Molecular weight was also estimated by sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis (8.5% slab gel) as described by Neville (23). The molecular weight was estimated by comparing the relative migration of purified hyaluronate lyase to the migration of bovine serum albumin (Armour), ovalbumin (Sigma), and lysozyme (Sigma). Relative migration was plotted against the log of molecular weight. Polyacrylamide gel electrophoresis. To demonstrate enzymatic activity after electrophoresis, polyacrylamide gels were run in an E-C vertical gel electrophoresis cell C 4 74 (St. Petersburg, Fla.). Both the 5% gel and the pH 7.0 buffer were prepared according to E-C technical bulletin no. 134. The electrophoresis buffer was modified by the addition of dithiothreitol (2 mM). After 3 h of pre-electrophoresis and a change of buffer, purified enzyme was electrophoresed at 120 mA for up to 9 h. The electrode buffer was constantly circulated, and the gel was maintained at 4 to 10°C to prevent enzyme inactivation. After electrophoresis, gels were stained for protein with 0.2% amido black 10B or were stained for carbohydrate according to the procedure of Fairbanks et al. (11). Duplicate gels were cut into 2.4-mm segments, mascerated in Sorensen's buffer (pH 6.0), and assayed for enzymatic activity after 12 h. Enzyme kinetics. The Michaelis constant was de-
728
HILL
INFECT. IMMUN.
termined for fraction IV enzyme with the tetrasaccharide of hyaluronate as substrate. The substrate concentration ranged from 0.074 to 2.4 mM tetrasaccharide in pH 6.0, 0.5 phosphate buffer. The reaction mixture was incubated for 1 h at 37°C, and the product was determined spectrophotometrically. The Km was determined from a Lineweaver-Burk reciprocal plot (10). Determination of other streptococcal products. Proteinase was determined by casein hydrolysis (7). Deoxyribonuclease (DNase) B (ADB) and streptolysin 0 (ASO) were determined with a commercial serological test kit (Beckman, Fullerton, Calif.). Nuclease activity was also determined by depolymerization of tritiated Escherichia coli deoxyribonucleic acid (DNA) obtained from N. Cozzarelli (Department of Biochemistry, University of Chicago). The labeled DNA had
a
specific activity of 2.3
x
108
,umol in pH 7.5, 30 ,iM tris(hydroxymethyl)aminomethane and 0.5 M ethylenediaminetetraacetic acid. The enzyme preparations were incubated for 2 h with the labeled DNA before chilling and the addition of carrier salmon testes DNA (Worthington). After thorough mixing, 2.5 M perchloric acid was added at 0°C and incubated for 10 min before removal of undigested DNA by centrifugation. A comparison was made between the nonprecipitable counts for the crude and purified enzyme preparations in toluene-based scintillation fluid. Streptokinase activity was determined with a (serological) kinase test kit from Fujizaki Pharmaceutical Co. (Tokyo, Japan). Hyaluronate lyase antisera. The fraction III enzyme was mixed with an equal volume of complete Freund adjuvant so that the final protein concentration was 0.06 mg/ml. The antigen was subcutaneously injected at two sites in 0.5-ml quantities into New Zealand White rabbits once a week over a 4week period before bleeding. Antihyaluronate lyase titer was determined with a Difco AHT kit and by a modification of the spectrophotometric assay for hyaluronic acid (13). In one row of tubes, 0.25 ml of immune serum was diluted from 1:32 to 1:2048. A duplicate row of tubes was made with serum from the corresponding rabbits before immunization. To each tube was added 0.25 ml of a 1:20 dilution of fraction III enzyme containing 1.2 mg of protein per ml. After 1 h of incubation at 37°C, 2.0 ml of a 0.2mg/ml, phosphate-buffered solution (pH 6.0) of hyaluronate was added to each tube and incubation was resumed for 10 min. The activity of free hyaluronate lyase was stopped by the addition of 0.5 ml of 20% perchloric acid at 0°C. The supernatant from each tube was decanted and the optical density was measured at 230 nm. AHT titer was recorded as the highest dilution of immune serum that had a lower optical density than normal serum. Immunodiffusion. The micro-Ouchterlony technique as described by Crowle (8) was used with 1.0% lonagar no. 2, buffered at pH 7.4 with 0.1 M phosphate. Human immune serum globulin was obtained from Hyland Laboratories (Costa Mesa, Calif.). Rabbit anti-hyaluronate lyase was prepared as described above. cpm per
RESULTS Production of hyaluronate lyase. More hyaluronate lyase was produced in veal infusion broth cultures than in Todd-Hewitt or brain heart infusion cultures. The dialyzable fraction of veal infusion broth as a medium yielded only 30% of the cells and 20% of the enzyme as the whole medium. Trypsinization of whole veal infusion broth resulted in a medium that yielded twice the hyaluronate lyase as the whole medium. In addition, trypsinization increased the efficiency of enzyme purification, especially at the gel filtration step. Cultures remained in log phase for 8 to 9 h. Peak enzyme production was during the 6th h and the enzyme level in cultures did not appreciably decline for 24 h. Agitation of cultures or the addition of 0.20% glucose increased cell densities, however, levels of detectable enzyme were reduced. Constant adjustment of the pH to 6.8 did not return hyaluronate lyase production to the normal level. The addition of 600 ug of hyaluronate or hyaluronate tetramer per ml increased total hyaluronate lyase activity by 40 and 20%, respectively (Table 1). Cultures containing these two substrates were uniformly less turbid at the end of log phase. The addition of hyaluronate dimer had a negligible effect on total enzymatic activity, whereas the addition of either N-acetylglucosamine or glucuronic acid reduced hyaluronate lyase activity by 40%. Enzyme purification. Ammonium sulfate precipitation and dialysis of the fraction I enTABLE 1. Hyaluronate lyase activation Relative OD600 of hyaluronConditioning substancea cultureb ate lyase yieldc 1.0 0.58 None 1.4 0.45 Hyaluronic acid 1.2 0.51 Tetramer 0.58 0.9 Dimer 0.6 0.58 N-acetylglucosamine 0.6 0.55 Glucuronic acid a Exponentially growing cells of strain 10402 were inoculated (1:100 dilution) into media containing one of each of the substrates. The substrates were present at 600 Ag/ml and were obtained as described in Materials and Methods. b Cultures were incubated to the end of log phase before cells were removed by centrifugation. OD, Optical density. c Relative amounts of hyaluronate lyase were determined spectrophotometrically in culture supernatants and after fractional ammonium sulfate precipitation.
STREPTOCOCCAL HYALURONATE LYASE
VOL. 14, 1976
729
TABLE 2. Purification of streptococcal hyaluronate lyase Fraction
Treatment
Total teinapro-
Enzyme Sp act uniac
I II III IV
None 0.32 kg 1.2 9.5 x 10-2 40-60% (NH4)2SO4 1.6 g 31.4 5.8 DEAE A-50 Sephadex 190 mg 317 48.8 Sephadex G-200 3.3 mg 323 248.5 a Protein determined according to Layne (17). b Hyaluronate lyase units were determined according to Greiling (13).
Punfication uifctin 0 1.2 x 102 9.1 X 102 1.2 x 104
Yield (% Yel
100 61 30 12
TABLE 3. Distribution of extracellular products in hyaluronate lyase preparations Prepne Product
Detection methoda
Crude
Hyaluronate lyase Streptokinase Streptolysin 0 DNase B
Pure
Serological
1:128 1:5,200 None Less than 40 1:40 None 1:160 Less than 1:40 DNA degradation 1 pmol/h per ,mI of DNA Negligible degradation Proteinase Casein digestion None None a Assay techniques are described in Materials and Methods. b Dilutions represent titration of hyaluronate lyase preparations against standard sera against other streptococcal products.
zyme reduced total protein to less than 1.0% of that in the crude broth. Seventy percent of the enzymatically active protein precipitated at 40 to 60% saturation and another 18% precipitated in the 60 to 80% saturation fraction. After each purification step, less enzyme was recovered by fractional precipitation (Table 2). When the protein concentration was below 200 ,ug/ml, ammonium sulfate precipitation could not be used for quantitative recovery of enzyme. Fraction III enzyme was obtained by ion-exchange chromatography of the dialyzed ammonium sulfate concentrates. On a DEAE'A-50 Sephadex column, about 80% of the enzymatically active protein eluted with 0.05 M phosphate at pH 6.0 (Fig. 1). The remaining enzyme eluted from the column with a sodium chloride gradient. Hyaluronate lyase eluting from the column before the salt gradient did not correspond to a protein peak, whereas the elution of enzyme with the NaCl salt gradient corresponded to the elution profile of protein. When the two hyaluronate lyase fractions were separately rechromatographed on DEAE A-50, they could not be distinguished. After ion-exchange chromatography, hyaluronate lyase was further purified on gel filtration columns. Hyaluronate lyase eluted from a preparative G-200 column as a single broad enzymatic peak; however, a distinct protein peak was not observed. Contaminants eluted after the enzyme as a single major peak. Enzymatically active fractions which were
pooled eluted as a single distinct protein peak when rechromatographed on a Sephadex G-150 column (Fig. 2). To obtain enzyme free of any detectable contaminants, fraction IV hyaluronate lyase was passed through a Sepharose 6B column. The enzyme was retarded on the column until contaminants had eluted. Determination of other streptococcal products. Fraction I enzyme contained appreciable amounts of DNase B but small amounts of streptokinase, streptolysin 0, and proteinase. Fraction IV enzyme was contaminated with only traces of DNase B, which were subsequently removed by Sepharose 6B purification. Gel diffusion on microscope slides revealed that crude hyaluronate lyase contained at least three antigens that were detectable with pooled human immune immunoglobulin G (Fig. 3). Specific rabbit antihyaluronate lyase also revealed more than one antigen, but the precipitin lines were indistinct. In contrast, neither antisera detected more than one antigen in purified enzyme preparations. AHTs of the specific rabbit antisera were determined with a commercial antihyaluronidase kit and by a spectrophotometric assay developed in this laboratory. Results of the enzyme blocking spectrophotometric assay were comparable to the commercial assay. One rabbit developed an AHT of 1,024 units, and two other animals developed antibody titers of 256 units. When the standard serum control in the commercial AHT kit was used, crude hyaluro-
730
INFECT. IMMUN.
HILL l150
~.140 130 -120 z
Io
-110 X
Go 0
s -80LU.
-ioo -
0 (.4
-90 0
0
- 70 60
z tu
I.Of 0
('
-
-50 it) 40 Z -30 0 ° 20 a
CL
10
FRACTIONS FIG. 1. DEAE-Sephadex A-50 chromatography of fraction II hyaluronate lyase. The first 40 fractions were eluted with 0.05 M phosphate (pH 6.0). Later fractions were eluted with a continuous salt gradient. Symbols: (0) Elution profile of proteins; (a) elution of enzymatic activity. The details of ion exchange are given in Materials and Methods. 130
.i12
.120
-~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ 910 Z'
.10
807
0.08
-~~~~~~~~~~~~~~~~70
OD
° Q .06
4
III
.
0
2
0
-60 u-, C.,
>
< ~~~~~~~~~~~50
-
LU U
14
16
18
20
22
24
26
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16
Z
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18
20
22
26
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28
30
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FRACTIONS
FIG. 2. Elution profile of fraction IV hyaluronate
lyase
Symbols: (0) Protein elution profile; (a) enzymatic activity.
nate lyase had to be diluted 1:128 before hyaluronate-protein complexes would form. The purified enzyme had to be diluted 1:5,200 (Table 3). Molecular weight determination. A calibrated Sephadex G-150 column was used to estimate the molecular weight of hyaluronate lyase. The enzyme had a Ka. that corresponded to a molecular weight of 70,000 on a selectivity curve (Fig. 4). SDS-polyacrylamide gel elec'tro-
on a
Sephadex G-150 column (2.5 by 33 cm).
phoresis was also used to estimate molecular weight. A single band was detected on an 8.5% slab gel which had a relative migration (Rf) corresponding to a molecular weight of about 50,000 on a calibration curve of known markers (Fig. 5). Polyacrylamide gel electrophoresis. Active hyaluronate lyase could be recovered from polyacrylamide gels only under the conditions de-
VOL. 14, 1976
STREPTOCOCCAL HYALURONATE LYASE
scribed in the Materials and Methods. The use of basic buffers, temperatures above 14°C, and no circulation of buffer between electrode chambers resulted in enzyme inactivation. It was also necessary for gels to be prerun and reduced. Under conditions that permitted the recovery of active enzyme, gels could be stained for both protein and carbohydrate at positions corresponding to enzymatic activity (Fig. 6). Kinetics. The Michaelis constant for fraction IV enzyme with hyaluronate tetramer as sub-
731
strate was 3.8 x 10-4 M (Fig. 7). The tetrasaccharide was prepared by the enzymatic digestion of potassium hyaluronate of an unknown molecular weight with hyaluronidase from sheep testes. Further digestion of the tetrasaccharide with streptococcal hyaluronate lyase results in equimolar amounts of N-acetyl-hyalobiouronic acid and an unsaturated derivative of this compound which characteristically was produced by hyaluronate lyases. Optimal pH and enzyme stability. The opti-
FIG. 3. Micro-Ouchterlony study of purified (well b) and crude (well e) hyaluronate lyase with specific rabbit antiserum (wells a and d) and human immune immunoglobulin G.
732
HILL
INFECT. IMMUN.
potentiated fraction I enzyme by three-fold, and reducing agents were also necessary to stabilize fraction IV enzyme.
500
400 300
2001 a 0 100
x
3:
90 80 70 60
50
40
30
201
se
I I
I
I |
I I
I
.1
.3
.4
.5
.6
.2
.7
.8
.9
DISCUSSION A strain of group A, type 4, S. pyogenes was grown in the presence of several forms of hyaluronic acid and its constituents to determine whether hyaluronate lyase activity would be affected. These results were consistent with the reports that streptococcal hyaluronate lyase is an inducible enzyme; however, it has not been ruled out that activity rather than enzyme production was influenced (13, 22). Data also suggested that one hydrolyzable substrate bond must be present for there to be enzyme stimulation. It was observed that hyaluronate tetrasaccharide stimulated enzyme activity but the dimer did not, even though streptococci reportedly metabolize the dimer (18). N-acetylglucosamine and glucuronic acid, the constituents of hyaluronic acid, depressed hyaluronate activity by 40%, suggesting that hyaluronate lyase is subject to repression as well as activation. Sallet al. (25) reported that streptococci meIJgman tabolize these monosaccharides more vigor1.0
Kcv
1001
FIG. 4. Selectivity curve for the estimation of molecular weight by the method of Andrews (5) with a Sephadex G-150 column. Points a through e represent aldolyase, hyaluronate lyase, ovalbumin, chymotrypsinogen, and ribonuclease, respectively.
so 70
a
60
mal pH for the purified enzyme was 6.0. Fraction I enzyme did not drop drastically in percentage of activity until pH 5.0; however, fraction IV enzyme became less active immediately below its optimum pH and was irreversibly inactivated in 30 min at pH 5.5. On the basic side of the optimal pH, the percentage of activity declined more gradually. At pH 6.8, fraction IV enzyme was only 40% as active as at the optimal pH, but the enzyme remained stable so that full activity was observed when the pH was adjusted to the optimum. At pH values above 7.0, both the crude and purified enzyme became unstable, and irreversible loss of activity was observed within 24 h. At pH 8.6 all enzymatic activity was irreversibly lost after 10
501
b
NO 40 x
3¢ 30 20~
d
min.
After cells were removed from a culture, frac.3 .4 .7 .6 .8 .2 tion I enzyme retained full activity for 1 week, RF at room temperature if reducing agents were FIG. 5. SDS-acrylamide gel electrophoretic estipresent. Fraction IV enzyme lost 50% of its mation of molecular weight. Points a, b, c, and d activity after 3 h at room temperature. Both the represent the respective migrations of bovine serum purified and crude enzyme retained full activity albumin, hyaluronate lyase, ovalbumin, and lysofor 2 years at -20°C. Addition of dithiothreitol zyme on an 8.5% slab gel (pH 8.5). .5
STREPTOCOCCAL HYALURONATE LYASE
VOL. 14, 1976
, ,
, , ,,1-,,, ., , ,= "IC E
z
0
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' 15 20
10
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MIGRATIONimm,
733
of this preparation in any way. Gel diffusion of fraction II enzyme revealed at least three antigens and SDS-gel electrophoresis revealed that up to 13 proteins were present. Although streptokinase and proteinase activities were eliminated by fractional ammonium sulfate precipitation, the resulting preparations were not pure. Fraction III enzyme is similar in preparation to the hyaluronate lyase described by Greiling et al. (13, 14). In agreement with these authors, it was observed that most of the enzyme eluted from ion-exchange columns with 0.05 NaH2PO4, but they also reported that a major protein peak contained the enzyme. In the current work, enzymatic activity was associated with a small fraction of the total protein (Fig. 1). A secondary enzymatic peak was associated with protein that eluted with a subsequent salt gradient. Greiling et al. (13, 14) reported heterogeneity in the elution of hyaluronate lyase, and they attributed this to multiple forms of the enzyme. Because the secondary enzymatic peak
6. Acrylamide gel electrophoresis of purified hyaluro. nate lyase on a 5% slab gel (pH 7.0). The top eluted with 0.05 NaH2PO4 when rechromatoof the fA gure (I) is enzymatic activity recovered from s. The bottom (II) is a densitometric scan of graphed, it seems most likely that there is a gel ) and carbohy- single form of the enzyme and the elution propduplicatte gels stained for protein ( -). (erties of it can be influenced by other proteins. * After purification by gel filtration, hyaluronate lyase eluted from Sephadex G-150 as a single enzymatically active protein peak, in contrast with staphylococcal hyaluronate lyase which eluted from Sephadex G-100 as multiple forms (2). At low ionic strengths the staphylococcal enzyme did not elute from Sephadex columns. This was not true of the streptococcal enzyme, where it was observed that elution was retarded on Sepharose 6B independently of Km 3.8 X 10-4 ionic strength. Sepharose 6B retention was utilized to remove contaminants from fraction IV , ,I i . , , ,I | , , , , _, slice
drate
22
20
18
16
14
12
10
0
2
4
6
8
10
12
14
16
I X io-3
s FIG. 7. Lineweaver-Burk plot for the determination oft 'he Michaelis constant ofpurified hyaluronate lyase w ith hyaluronate tetrasaccharide as substrate.
enzyme.
After purification of hyaluronate lyase by gel filtration, single lines of precipitation were formed with antisera in gel diffusion studies. In agreement with Halbert and Auerbach (15), human immune serum gamma globulin was
more effective in the detection of precipitating streptococcal antigens than specific antisera prepared in rabbits. In one case, specific rabbit oxidatiion influence the activity of hyaluronate antiserum detected one antigen in fraction II enzyme, whereas human gamma globulin lyase vvithout affecting its synthesis. Fraction II enzyme is comparable to the hy- formed four lines of precipitation against the aluron;ate lyase purified by Mogilevskii (22). same antigen. Polyacrylamide gel electrophoresis revealed After ammonium sulfate fractionation, his prepar;ations were treated with cholesterol to that a single protein species was present in purified enzyme preparations. Carbohydrate removEestreptolysin 0. Fraction II preparations contairied a small amount of streptolysin 0 and staining suggested that streptococcal hyalurolarge a mounts of DNase. Treatment with cho- nate lyase is a glycoprotein. Although no other lesterol1 did not alter the antigenic composition bacterial hyaluronate lyase has been character-
ously t;han hyaluronate oligosaccharide. It has not be en ruled out that the products of this
734
HILL
INFECT. IMMUN.
ized to this degree, it has been shown that testicular hyaluronidase is a glycoprotein with a molecular weight of 61,000 by gel filtration methods (6). The molecular weight of the streptococcal hyaluronate lyase was about 70,000 by gel filtration and 50,000 by SDS-acrylamide electrophoresis. No other molecular weight estimates for hyaluronate lyase are available for comparison; however, molecular weight determinations for testicular hyaluronidase range from 43,000 to 126,000, depending on enzyme purity and the method of determination (6). The Michaelis constant for fraction IV enzyme was 3.8 x 10-4 M when measured with a homogeneous substrate of a known molecular weight. Greiling et al. (14) reported Km values of 3.2 x 10-2 and 1.3 x 10-2 for streptococcal hyaluronate lyase resolved by ion-exchange chromatography. The difference between his two Km values is not great enough to indicate that two different enzymes having two different Km values were studied. Differences in purity alone would cause variations in Km of this magnitude because of different efficiencies of substrate binding. The difference between the Km reported here and those of Greiling is probably due to his use of an undefined oligosaccharide as substrate. The earlier values were obtained with the use of hyaluronic acid of an undesignated molecular weight, whereas a homogeneous substrate of a known molecular weight was used in this study. Abramson and Friedman (1-3) resolved three to four forms of staphylococcal hyaluronate lyase by chromatographic and electrophoretic techniques. This quality of staphylococcal hyaluronate lyase became significant to the study of infectious disease with the contention that a correlation between enzyme form and the clinical manifestations of staphylococcal disease could be observed. Greiling et al. (14) reported three hyaluronate lyases from staphylococci and two from streptococci. The results reported here indicate that ATCC strain 10402, a throat isolate, produced a single hyaluronate lyase as determined by chromatographic, electrophoretic, and serological techniques. This does not rule out the possibility that other streptococci produce multiple hyaluronate lyases. ACKNOWLEDGMENTS I wish to express my appreciation to William Martin for guidance and the use of his laboratory facilities. I also wish to thank Eugene Fox for his critical evaluation of this manuscript and V. Ormiste, R. Lewert, and M. Yogore for technical assistance. This investigation was supported by United States Public Health Service grant GM603 from the National Institute of General Medical Sciences.
LITERATURE CITED 1. Abramson, C. 1967. Staphylococcal hyaluronate lyase: multiple electrophoretic and chromatographic forms. Arch. Biochem. Biophys. 121:103-106. 2. Abramson, C. 1974. Staphylococcal hyaluronidase isoenzyme profiles related to staphylococcal disease. Ann. N.Y. Acad. Sci. 236:495-507. 3. Abramson, C., and H. Friedman. 1968. Staphylococcal hyaluronate lyase: purification and characterization studies. J. Bacteriol. 96:886-892. 4. Abramson, C., and H. Friedman. 1969. Electrophoretic characteristics of staphylococcal hyaluronate lyase. J. Bacteriol. 97:715-718. 5. Andrews, P. 1964. Estimation of molecular weights of proteins by Sephadex gel filtration. Biochem. J. 91:222-233. 6. Borders, C. P., and M. A. Raftery. 1968. Purification and partial characterization of testicular hyaluronidase. J. Biol. Chem. 243:3756-3762. 7. Cohen, J. 0. 1969. Effect of culture medium components and pH on the production of M protein and proteinase by group A streptococci. J. Bacteriol. 99:737-744. 8. Crowle, A. J. 1958. A simplified micro-double diffusion agar precipitin technique. J. Lab. Clin. Med. 52:784787. 9. Davidson, E. A. 1966. Analysis of sugars found in mucopolysaccharides, p. 54. In S. P. Colowich and N. 0. Kaplan (ed.), Methods in enzymology, vol. 8. Academic Press Inc., New York. 10. Dawes, E. A. 1969. Quantitative problems in biochemistry, 4th ed. The Williams and Wilkins Co., Baltimore. 11. Fairbanks, G., T. L. Steck, and F. H. Wallach. 1971. Electrophoretic analysis of the main polypeptides of the human erythrocyte membrane. Biochemistry 10:2606-2617. 12. Ginsberg, I. 1972. Mechanism of cell and tissue injury induced by group A streptococci: relation to poststreptococcal sequele. J. Infect. Dis. 126:294-340. 13. Greiling, H. 1963. Hyaluronic acid, p. 87-90. In H. Bergmeyer (ed.), Methods of enzymatic analysis. Academic Press, Inc., New York. 14. Greiling, H., H. W. Stuhlsatz, and T. Eberhard. 1965. Zur heterogenitat der hyaluronatlyase. Z. Physiol. Chem. 340:243-248. 15. Halbert, S. P., and T. Auerbach. 1961. The use of precipitin analysis in agar for the study of human streptococcal infections. IV. Further observations on the purification of group A extracellular antigens. J. Exp. Med. 113:131-157. 16. Kass, E. H., and C. V. Seastone. 1944. The role of the mucoid polysaccharide (hyaluronic acid) in the virulence of group A hemolytic streptococci. J. Exp. Med. 79:319-329. 17. Layne, E. 1957. Spectrophotometric and turbidimetric methods for measuring proteins, p. 451-454. In S. P. Colowich and N. 0. Kaplan (ed.), Methods in enzymology, vol. 3. Academic Press Inc., New York. 18. Linker, A., K. Meyer, and P. Hoffman. 1956. The production of unsaturated uronides by bacterial hyaluronidases. J. Biol. Chem. 219:13-25. 19. Ludowieg, J., B. Vennesland, and A. Dorfman. 1961. The mechanism of action of hyaluronidases. J. Biol. Chem. 236:333-339. 20. McCarty, M. 1973. Streptococci, p. 708-726. In B. D. Davis, R. Dulbecco, H. N. Eisen, H. S. Ginsberg, W. B. Wood, and M. McCarty (ed.), Microbiology, 2nd ed. Harper and Row, Hagerstown, Md. 21. Meyer, K., and M. M. Rapport. 1952. Hyaluronidases, p. 199-236. In F. F. Nord (ed.), Advances in enzymology, vol. 8. Interscience Pub., New York.
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22. Mogilevskii, M. Sh. 1964. Isolation and purification of streptococcal hyaluronidase. Fed. PIfoc. 23:T559-561. 23. Neville, D. M. 1971. Molecular weight determination of protein-dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system. J. Biol. Chem. 246:6328-6334. 24. Pike, R. M. 1948. Streptococcal hyaluronic acid and
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hyaluronidase. I. Hyaluronidase activity of noncapsulated group A streptococci. J. Infect. Dis. 83:1-11. 25. Sallman, B., J. M. Birkeland, and C. T. Gray. 1951. Hyaluronic acid utilized by hemolytic streptococci in relation to possible hyaluronidase function in pathogenesis. Proc. Soc. Exp. Biol. Med. 76:467-471.
