- Email: [email protected]
Isolation and Characterization of Hyaluronidase from Streptococcus uberis
Zbl. Bakt. 271, 46-53 (1989)
Isolation and Characterization of Hyaluronidase from Streptococcus uberis P. SCHAUFUSS, R. STING, W. SCHAEG, and H. BLOBEL Institut fur Bakteriologie und Immunologie, Fachbereich Veteriniirmedizin, Justus-LiebigUniversitiit Giegen, D-6300 Giegen
With 8 Figures· Received August 29, 1988 . Accepted November 23, 1988
Abstract All tested cultures of Streptococcus uberis produced free hyaluronidase. Hyaluronidase could be isolated by ammonium sulfate precipitation and was further purified by chromatography on DEAE-cellulose, gelfiltration on ultragel ACA44 and isoelectric focussing. The purification factor was estimated to be 1689. The purified hyaluronidase had an isoelectric point at pH 4.9 and a molecular weight of approximately 54000 D. It showed maximal enzyme activity at pH 6.0 and 45°C. The Michaelis constant was estimated to be 7.0 X 10-2 mglml. Hyaluronidase activity was stimulated by Ca++, Mg++, Mn++, Co++, Li+, and K+ and inhibited by Zn++ and Cd++ at final concentrations of 10 mmolll, respectively. Zusammenfassung AIle untersuchten Streptococcus uberis-Kulturen produzierten Hyaluronidase. Hyaluronidase konnte durch Ammoniumsulfatfiillung des Kulturiiberstandes, Ionenaustauschchromatographie (DEAE-Cellulose), Gelfiltration (Ultragel AC~4) und isoelektrischer Fokussierung gereinigt werden. Der Reinigungsfaktor betrug 1689. Die gereinigte Hyaluronidase hatte einen isoelektrischen Punkt von pH 4,9 und ein Molekulargewicht von ungefiihr 54000 D. Maximale Enzymaktivitiiten wurden bei pH 6,0 und 45°C ermittelt. Die Michaelis-Konstante betrug 7,0 X 10-2 mglml. Die Hyaluronidase-Aktivitiit wurde durch Ca++, Mg++, Mn++, Co++, Li+ und K+ stimuliert und durch Zn++ und Cd++ in einer Endkonzentration von jeweils 10 mmolll gehemmt.
Introduction Streptococci belonging to serological groups A, B, C, G, H, L, Streptococcus mitis (10) and S. salivarius (6) are known to secrete hyaluronidase into the culture medium. Hyaluronidase depolymerizes hyaluronic acid, which is an essential component of connective tissues. Thus, production of hyaluronidase might facilitate dissemination of streptococci into mucous membranes (5). Among streptococci S. uberis is a common
Hyaluronidase from Streptococcus uberis
47
pathogen known to cause mastitis in cattle. Production of hyaluronidase by S. uberis could facilitate the penetration of these bacteria into the tissue of the mammary gland and therefore playa role in pathogenicity. In the present study hyaluronidase production from S. uberis was analyzed and the purified enzyme characterized. The present findings could be of some relevance for pathogenic studies with S. uberis.
Materials
and Methods
Streptococci. All 16 S. uberis-cultures had been isolated from bovines and were identified as described by Hahn et a. (6). They included 3 strains (CCM 5673, 5674 and 6186) from the Czechoslovak Collection of Microorganisms. In addition, 1 hyaluronidase-positive culture of S. pyogenes (serological group A) and 1 hyaluronidase-negative culture of S. bovis (serological group D) served as controls. Enzyme assays. The S. uberis-cultures were screened for hyaluronidase-activity on hyaluronidase-test-agar (17). The substrate-solution contained 50 mg hyaluronic acid (HA) (Sigma, Deisenhofen, FRG) and 1.25 g bovine serum albumin (Serva, Heidelberg, FRG) in 25 ml aqua dest. Hyaluronidase-activity was also determined quantitatively photometrically (2). For this 10 tAl of a sample to be tested for enzyme activity was added to a substratesolution consisting of 5 fA-g HA in 90 tAl 20 mmol/l sodium acetate with 10 mmol/l NaCI at pH 6.0. After 1 h at 37°C the enzyme-reactions were terminated by the addition to each tube of 1 ml aqua dest. at 4°C. Then 900 fA-I carbocyanine-dye-solution (Carbocyanine-dye from Sigma) (2) were added and the optical density (OD) at 640 nm measured. The blanks contained 10 tAl of boiled (10 min) hyaluronidase-solution, 5 fA-g hyaluronic acid, 90 tAl reaction buffer, 1 ml aqua dest. and 900 fA-I dye-solution. One unit (u) of hyaluronidase split 1 mg HA within 1 h. Purification of s. uberis-hyaluronidase. s. uberis strain CCM 6186 had the highest hyaluronidase-activity and was therefore used for production of the enzyme. The streptococci were incubated for 18 h at 37°C in 2 I brain heart infusion (BHI, Merck, Darmstadt, FRG) , then removed by centrifugation (20 min, 12500 x g) and subsequent filtration (average pore diameter of 0.65 f.tffi, Millipore, Neu-Isenburg, FRG). Hyaluronidase was precipitated from the cell-free culture supernatant by the addition of ammonium sulfate at a final concentration of 60%. The precipitate was collected by centrifugation (30 min, 12500 X g), dissolved in 20 milO mmol/l phosphate buffer, pH 6.8 and dialyzed against this buffer for 48 h at 4°C. The hyaluronidase-solution was then applied to a DEAE-cellulose column (3 x 10 cm, DE-52, Whatman, Biochemical Ltd., Maidstone, England) equilibrated with 10 mmol/l phosphate buffer, pH 6.8. The column was subsequently washed with the phosphate buffer and hyaluronidase eluted with a linear gradient of 0 to 0.5 mOl/l NaCl at a flow rate of 40 mllh. Fractions with the highest hyaluronidase-activity were pooled and subjected to filtration on ultragel ACA44 column of 2.0 x 100 cm (LKB, Bromma, Sweden), equilibrated with 10 mmol/l phosphate buffer with 0.1 molll NaCI at pH 6.8. The column was loaded with 2 ml of the chromatographically purified hyaluronidase-solution. Hyaluronidase was then eluted with the same buffer at 10 mllh. Fractions with highest hyaluronidase-activity were pooled again and applied to an isoelectric focussing column (LKB) under the conditions described by Schaeg et al. (15). The carrier ampholytes ranged from pH 3.5 to 10. SDS-PAGE. Samples containing approximately 2 fA-g protein were subjected to SDSpolyacrylamide-gel-electrophoresis (SDS-PAGE) as described by Laemmli (11) for 4 h at 30 rnA and 120 V. Then the gel was silver-stained according to Heukeshoven and Dernick (8). Standardized markers (Sigma) served for determinations of molecular weights (19). Protein determinations. Protein was precipitated from the hyaluronidase-solutions by the addition of an equal volume of 20% (w/v) trichloroacetic acid (TCA). After 16 h at 4°C precipitates were centrifugated and washed 2 X in 10% TCA. Protein content was measured by the method of Lowry et al. (13) with bovine serum albumin (Serva) as a standard.
48
P. Schaufuss, R. Sting, W. Schaeg, and H. Blobel
Enzyme kinetics. The Michaelis constant (Km) was determinated using 10 III of purified S. uberis-hyaluronidase and varying concentrations (0.03-0.1 mg/ml) of HA in the hyaluronidase-assay described above. Velocity of the enzyme reaction was expressed as mg HA split after 30 min at 37°C. Double-reciprocal plots of velocity versus HA-concentrations according to Lineweaver and Burk (12) served to determine Km-values. The method of "least squares" was used to find the slope of the straight line. Enzyme characterizations. Hyaluronidase-activity was measured in 20 mmolll sodium acetate with 10 mmolll NaCI at pH-values from 4.0 to 7.0. Subsequently, hyaluronidaseactivity at pH 6.0 was determined after respectively 30 min at various temperatures between 30 and 60°C. Hyaluronidase-activity was also evaluated after addition of various cations at respectively 10 mmol/l to the assay mixture on the basis of hyaluronidase-activity in the unsupplemented control. Results All 16 S. uheris cultures produced free hyaluronidase as indicated by the development of clear zones surrounding streptococcal growth on hyaluronidase-test-agarplates. S. uheris strain CCM 6186 revealed highest hyaluronidase activity and was therefore selected for the isolation of the enzyme. Hyaluronidase could be isolated from the cell-free culture supernatant of this strain in a highly purified form by a series of purification steps (Table 1). Precipitation of hyaluronidase with ammonium sulfate at a final concentration of 60% increased the specific activity from 0.6 u/mg to 6.7 ulmg. Further purification was achieved by chromatography on DEAE-cellulose. Most of the enzyme was eluted from the adsorbent column with NaCI at concentrations between 0.07 and 0.1 molll (Fig. 1). Following gelfiltration on ultragel AC4 increased further the specific hyaluronidase activity to 390.5 ulmg (Fig. 2). Finally, isoelectric focussing (Fig. 3) yielded a homogeneous preparation of highly purified hyaluronidase with an isoelectric point of pH 4.9 and a molecular weight of 54000 D (Fig. 4). The purified enzyme exhibited maximal activity at pH 6.0 (Fig. 5) and 45°C (Fig. 6). The Michaelis constant was estimated to be 7.0 X 10-2 mg/ml (Fig. 7). Hyaluronidase activity increased in the presence of Ca++, Mg++, Mn++, Co++, Li+, and K+ for 40-50% and decreased with Zn++ and Cd++ for 30-40% at a final concentration of 10 mmolll respectively (Fig. 8).
Table 1. Purification of hyaluronidase from S. uberis, strain CCM 6186 Step of purification (u/mg)*
Specific activity factor
Purification
Culture supernatant (NH4hS04 precipitation DEAE-cellulose Gel-filtration Isoelectric focussing
0.6 6.7 196.4 390.5 1013.5
1 11.2 327.3 650.8 1689.2
* units hyaluronidase per mg protein
Hyaluronidase from Streptococcus uberis
-
49
E
::J
-:>.
>
u ttl
125
0.15
100 0.10
75
0
E
III
ttl "0
50
0.05
c:
0
to.
::J
25
U ttl Z
ttl
>-
X
5
10 Fractions
0.00
15
Fig. 1. Chromatography of hyaluronidase from S. uberis strain CCM 6186 on DEAEcellulose. Hyaluronidase was eluted with 10 mmolll phosphate buffer pH 6.8, containing NaCI at a linear gradient of 0.0 to 0.5 molll. NaCI (-), hyaluronidase-activity (A)
OD 280
E
0.15
::J
>
10 0.10
u
ttl
III
ttl "0
5
0.05
c:
oto.
::J
ttl :>. X
27
30
40
50
0.00
Fractions
Fig. 2. Gelfiltration of chromatographically purified hyaluronidase from S. uberis strain CCM 6186 on ultragel ACA44 • Hyaluronidase-activity (A), OD 280 (x- - - -x) 4 Zbl. Bakt. 27111
50
P. Schaufuss, R. Sting, W. Schaeg, and H. Blobel
~
l 3
,
E ::J
..
20
~
15
>>
to
QI 1/1
to
"0
10
c
~
::J
It
5.5 pH
5
iii
• 0.2
0.0
4.5
>-
:I:
6 7
5.0
0.1
18 20
25
30
Fractions
Fig. 3. Isoelectric focussing of hyaluronidase from S. uberis strain CCM 6186. Hyaluronidase activity (.A), OD Z80 (x- - - -x), pH (e) Fig. 4. Estimation of molecular weight of purified hyaluronidase from S. uberis strain CCM 6186 by SDS-PAGE. Bands 1, 2, 3, 4, 5, 6 and 7 represented uz-macroglobulin (180 kD), j3-gaiactosidase (116 kD), fructose-6-phosphate kinase (84 kD), pyruvate kinase (58 kD), fumarase (48 kD), lactate dehydrogenase (36.5 kD), and triosephosphate isomerase (26.6 kD). Approximately 2 Ilg of the purified hyaluronidase was loaded on the gel.
1.0 1.0
-. >-
>.-
>
>
uco QI
u
1\1
0.5
0.5
>
>
iii
iii
Ii ...
~
5.0
6.0
7.0
30
pH Fig. 5. Influence of pH on hyaluronidase activity (.A) Fig. 6. Influence of temperature on hyaluronidase activity (.A)
40
50
Temperature (·C )
60
Hyaluronidase from Streptococcus uberis
51
O.S
0.4 0.3 >-
0.2
.~
0.1
ti co CI> >
OO+-JLl-"...l-lL.I.--"'-..L.....JL.L....IL.L....f:';';-"':';' •
:; 0.1 CI>
.. 0.2 0.3 0.4 0.5
Fig. 7. Influence of Ca++, Mg++, Mn++, Co++, K+, Li+, Zn++ and Cd++ on hyaluronidase activity. Hyaluronidase activities with addition of cations were compared with hyaluronidase activity of the standard assay (0.0.)
1
V
60
40
20
-20
-10
10
20
30
40
1
[S] Fig. 8. Reciprocal (Lineweaver-Burk) plot of purified hyaluronidase from S. uberis strain CCM 6186. Using varying amounts of hyaluronic acid velocity of enzyme reaction (v) was expressed as mg hyaluronic acid split after incubation for 30 min at 37°C. [S] = mg hyaluronic acid/ml, hyaluronidase activity (.A)
52
P. Schaufuss, R. Sting, W. Schaeg, and H. Blobel Discussion
Hyaluronidase activities have been detected in culture media of streptococci belonging to serological groups A, B, C, G, H, L, in those of Streptococcus mitis (10) and s. salivarius (6). The enzyme, which was mainly produced during the logarithmic growth phase (4), depolymerized hyaluronic acid (14). Hyaluronidase, thus, could facilitate the dissemination of streptococci into mucous membranes or other tissues (5). It was, therefore, called "spreading factor" (3). Our studies revealed that S. uberis produced this enzyme in significant concentrations. Hyaluronidase could be purified to a homogeneous protein by precipitation with ammonium sulfate, chromatography on DEAE-cellulose, gelfiltration on AC~4 ultragel and isoelectric focussing. This was in agreement with the findings of Schaufup et al. (16) on hyaluronidase from S. equi and S. pyogenes (9). On the other hand, Abramson (1) reported that staphylococcal hyaluronidase consisted of multiple electrophoretic and chromatographic forms. The purified hyaluronidase from S. uberis had a molecular weight of approximately 54000 D as estimated by SDS-PAGE. A similar molecular weight had been determined for hyaluronidase from S. equi by Schaufup et al. (16) and for hyaluronidase from S. pyogenes (9). In contrast Gerlach and Kohler (10) found a molecular weight of 75000 D for hyaluronidase from S. pyogenes. These differences could possibly be explained by different methods of molecular weight determination. Purification of hyaluronidase by isoelectric focussing indicated an isoelectric point (PI) of pH 4.9. Schaufup et al. (16) reported an PI of pH 5 for hyaluronidase from S. equi. Smyth and Fehrenbach (18) described isoelectric points (PIs) of pH 4.4 and 4.3 for hyaluronidase preparations from streptococci of serological groups A and C respectively. Hallas and Widdowson (7) found PIs at pH 5.9, 4.5 and 4.6 for hyaluronidase from streptococci of serological groups A, C and G. In the present study a pH-optimum of 6 and a temperature-optimum of 45°C was found to be optimal for hyaluronidase activity of S. uberis. Similar datas were obtained for hyaluronidase of S. equi (16). The Michaelis constant (KM) of purified hyaluronidase from S. uberis was 7.0 x 10-2 mg/ml. Schaufup et al. (16) reported KM-values of 6.2 x 10-2 mg/ml for hyaluronidase of S. equi. Hill (9) described KM-values for S. pyogenes of 3.8 X 10-4 molll and Gerlach and Kohler (4) of 4.0 X 10-2 mg/ml. These present findings about hy:tluronidase production of S. uberis and the characterization of the purified enzyme could be of interest for further studies on pathogenicity of these streptococcal species. References 1. Abramson, c.: Staphylococcal hyaluronate lyase: Multiple electrophoretic and chromatographic forms. Arch. Biochem. Biophys. 121 (1967) 103-106 2. Benchetrit, L. c., S. L. Pahuja, E. D. Gray, and R. D. Edstrom: A sensitive method for the assay of hyaluronidase activity. Anal. Biochem. 79 (1977) 431-437 3. Duran-Reynals, F.: Tissue permeability and spreading factors in infection. Bact. Rev. 6 (1942) 197-252 4. Gerlach, D. and W. Kohler: Hyaluronate lyase from Streptococcus pyogenes. I. The production and isolation of hyaluronate lyase. Zbl. Bakt. Hyg., I. Abt. Orig. A 221 (1961) 333-339 5. Ginsburg, I.: Mechanism of cell and tissue injury induced by group A streptococci: Relation to poststreptococcal sequelae. J. Infect. Dis. 126 (1972) 294--340
Hyaluronidase from Streptococcus uberis
53
6. Hahn, G., W. Heeschen und H. Tolle: "Streptococcus". Eine Studie zur Struktur,
Biochemie, Kultur und Klassifizierung. Kiel. Milchw. Forschungsber. 22 (1970) 333-546 7. Hallas, G. and j. P. Widdowson: Antibody to hyaluronidase of streptococci of Lancefield groups C and G. In: S. E. Holm and P. Christensen (eds.), Basic Concepts of Streptococcal Diseases. Reedbooks Ltd., Chertsey, Surrey (1981) 8. Heukeshoven, J. and R. Dernick: Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 6 (1985) 103-112 9. Hill, j.: Purification and properties of streptococcal hyaluronate lyase. Infect. Immun. 14 (1976) 726-735 10. Kohler, W.: Die Serologie des Rheumatismus und der Streptokokkeninfektionen, 3. Auf!.]. A. Bart Verlag, Leipzig (1963) 11. Laemmli, W. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4 • Nature 227 (1970) 68~85 12. Lineweaver, H. and D. Burk: The determination of enzyme dissociation constants. ]. Am. Chern. Soc. 56 (1934) 658-666 13. Lowry, O. H., N.J. Rosebrough, A. 1. Farr, and R. J. Randall: Protein measurement with the Folin phenol reagent. J. BioI. Chern. 193 (1951) 265-275 14. Ludowieg, j., B. Vennesland, and A. Dorfman: The mechanism of action of hyaluronidases. ]. BioI. Chern. 236 (1961) 333-339 15. Schaeg, W., R. Bingol, and H. Blobel: Purification of penicillinase (~-lactamase) and acid phosphatase from Staphylococcus aureus in one procedure. Biochim. Biophys. Acta 268 (1972) 542-549 16. Schaufup, P., R. Sting, and H. Blobel: Purification and characterization of hyaluronidase from Streptococcus equi. X'h Lancefield International Symposium on Streptococci and Streptococcal Diseases, Cologne, FRG, 1.-4. Sept. 1987 17. Smith, R. F. and N. P. Willet: Rapid plate method for screening hyaluronidase and chondroitin sulfatase-producing microorganisms. Appl. Microbiol. 16 (1968) 1434-1436 18. Smyth, C. j. and F. J. Fehrenbach: Isoelectric analysis of haemolysins and enzymes from streptococci of groups A, C and G. Acta path. microbiol. scand. Sect. B 82 (1974) 860-870 19. Weber, K. and M. Osborn: The reliability of molecular weight determinations by dodecylsulfate-polyacrylamide gel electrophoresis. J. BioI. Chern. 244 (1969) 4406-4412 Dr. Peter Schaufup, Inst. f. Bakteriologie und Immunologie, Fachbereich Veterinarmedizin der Universitat Giel~en, Frankfurter Str. 107, D-6300 Giel~en
Isolation and Characterization of Hyaluronidase from Streptococcus uberis P. SCHAUFUSS, R. STING, W. SCHAEG, and H. BLOBEL Institut fur Bakteriologie und Immunologie, Fachbereich Veteriniirmedizin, Justus-LiebigUniversitiit Giegen, D-6300 Giegen
With 8 Figures· Received August 29, 1988 . Accepted November 23, 1988
Abstract All tested cultures of Streptococcus uberis produced free hyaluronidase. Hyaluronidase could be isolated by ammonium sulfate precipitation and was further purified by chromatography on DEAE-cellulose, gelfiltration on ultragel ACA44 and isoelectric focussing. The purification factor was estimated to be 1689. The purified hyaluronidase had an isoelectric point at pH 4.9 and a molecular weight of approximately 54000 D. It showed maximal enzyme activity at pH 6.0 and 45°C. The Michaelis constant was estimated to be 7.0 X 10-2 mglml. Hyaluronidase activity was stimulated by Ca++, Mg++, Mn++, Co++, Li+, and K+ and inhibited by Zn++ and Cd++ at final concentrations of 10 mmolll, respectively. Zusammenfassung AIle untersuchten Streptococcus uberis-Kulturen produzierten Hyaluronidase. Hyaluronidase konnte durch Ammoniumsulfatfiillung des Kulturiiberstandes, Ionenaustauschchromatographie (DEAE-Cellulose), Gelfiltration (Ultragel AC~4) und isoelektrischer Fokussierung gereinigt werden. Der Reinigungsfaktor betrug 1689. Die gereinigte Hyaluronidase hatte einen isoelektrischen Punkt von pH 4,9 und ein Molekulargewicht von ungefiihr 54000 D. Maximale Enzymaktivitiiten wurden bei pH 6,0 und 45°C ermittelt. Die Michaelis-Konstante betrug 7,0 X 10-2 mglml. Die Hyaluronidase-Aktivitiit wurde durch Ca++, Mg++, Mn++, Co++, Li+ und K+ stimuliert und durch Zn++ und Cd++ in einer Endkonzentration von jeweils 10 mmolll gehemmt.
Introduction Streptococci belonging to serological groups A, B, C, G, H, L, Streptococcus mitis (10) and S. salivarius (6) are known to secrete hyaluronidase into the culture medium. Hyaluronidase depolymerizes hyaluronic acid, which is an essential component of connective tissues. Thus, production of hyaluronidase might facilitate dissemination of streptococci into mucous membranes (5). Among streptococci S. uberis is a common
Hyaluronidase from Streptococcus uberis
47
pathogen known to cause mastitis in cattle. Production of hyaluronidase by S. uberis could facilitate the penetration of these bacteria into the tissue of the mammary gland and therefore playa role in pathogenicity. In the present study hyaluronidase production from S. uberis was analyzed and the purified enzyme characterized. The present findings could be of some relevance for pathogenic studies with S. uberis.
Materials
and Methods
Streptococci. All 16 S. uberis-cultures had been isolated from bovines and were identified as described by Hahn et a. (6). They included 3 strains (CCM 5673, 5674 and 6186) from the Czechoslovak Collection of Microorganisms. In addition, 1 hyaluronidase-positive culture of S. pyogenes (serological group A) and 1 hyaluronidase-negative culture of S. bovis (serological group D) served as controls. Enzyme assays. The S. uberis-cultures were screened for hyaluronidase-activity on hyaluronidase-test-agar (17). The substrate-solution contained 50 mg hyaluronic acid (HA) (Sigma, Deisenhofen, FRG) and 1.25 g bovine serum albumin (Serva, Heidelberg, FRG) in 25 ml aqua dest. Hyaluronidase-activity was also determined quantitatively photometrically (2). For this 10 tAl of a sample to be tested for enzyme activity was added to a substratesolution consisting of 5 fA-g HA in 90 tAl 20 mmol/l sodium acetate with 10 mmol/l NaCI at pH 6.0. After 1 h at 37°C the enzyme-reactions were terminated by the addition to each tube of 1 ml aqua dest. at 4°C. Then 900 fA-I carbocyanine-dye-solution (Carbocyanine-dye from Sigma) (2) were added and the optical density (OD) at 640 nm measured. The blanks contained 10 tAl of boiled (10 min) hyaluronidase-solution, 5 fA-g hyaluronic acid, 90 tAl reaction buffer, 1 ml aqua dest. and 900 fA-I dye-solution. One unit (u) of hyaluronidase split 1 mg HA within 1 h. Purification of s. uberis-hyaluronidase. s. uberis strain CCM 6186 had the highest hyaluronidase-activity and was therefore used for production of the enzyme. The streptococci were incubated for 18 h at 37°C in 2 I brain heart infusion (BHI, Merck, Darmstadt, FRG) , then removed by centrifugation (20 min, 12500 x g) and subsequent filtration (average pore diameter of 0.65 f.tffi, Millipore, Neu-Isenburg, FRG). Hyaluronidase was precipitated from the cell-free culture supernatant by the addition of ammonium sulfate at a final concentration of 60%. The precipitate was collected by centrifugation (30 min, 12500 X g), dissolved in 20 milO mmol/l phosphate buffer, pH 6.8 and dialyzed against this buffer for 48 h at 4°C. The hyaluronidase-solution was then applied to a DEAE-cellulose column (3 x 10 cm, DE-52, Whatman, Biochemical Ltd., Maidstone, England) equilibrated with 10 mmol/l phosphate buffer, pH 6.8. The column was subsequently washed with the phosphate buffer and hyaluronidase eluted with a linear gradient of 0 to 0.5 mOl/l NaCl at a flow rate of 40 mllh. Fractions with the highest hyaluronidase-activity were pooled and subjected to filtration on ultragel ACA44 column of 2.0 x 100 cm (LKB, Bromma, Sweden), equilibrated with 10 mmol/l phosphate buffer with 0.1 molll NaCI at pH 6.8. The column was loaded with 2 ml of the chromatographically purified hyaluronidase-solution. Hyaluronidase was then eluted with the same buffer at 10 mllh. Fractions with highest hyaluronidase-activity were pooled again and applied to an isoelectric focussing column (LKB) under the conditions described by Schaeg et al. (15). The carrier ampholytes ranged from pH 3.5 to 10. SDS-PAGE. Samples containing approximately 2 fA-g protein were subjected to SDSpolyacrylamide-gel-electrophoresis (SDS-PAGE) as described by Laemmli (11) for 4 h at 30 rnA and 120 V. Then the gel was silver-stained according to Heukeshoven and Dernick (8). Standardized markers (Sigma) served for determinations of molecular weights (19). Protein determinations. Protein was precipitated from the hyaluronidase-solutions by the addition of an equal volume of 20% (w/v) trichloroacetic acid (TCA). After 16 h at 4°C precipitates were centrifugated and washed 2 X in 10% TCA. Protein content was measured by the method of Lowry et al. (13) with bovine serum albumin (Serva) as a standard.
48
P. Schaufuss, R. Sting, W. Schaeg, and H. Blobel
Enzyme kinetics. The Michaelis constant (Km) was determinated using 10 III of purified S. uberis-hyaluronidase and varying concentrations (0.03-0.1 mg/ml) of HA in the hyaluronidase-assay described above. Velocity of the enzyme reaction was expressed as mg HA split after 30 min at 37°C. Double-reciprocal plots of velocity versus HA-concentrations according to Lineweaver and Burk (12) served to determine Km-values. The method of "least squares" was used to find the slope of the straight line. Enzyme characterizations. Hyaluronidase-activity was measured in 20 mmolll sodium acetate with 10 mmolll NaCI at pH-values from 4.0 to 7.0. Subsequently, hyaluronidaseactivity at pH 6.0 was determined after respectively 30 min at various temperatures between 30 and 60°C. Hyaluronidase-activity was also evaluated after addition of various cations at respectively 10 mmol/l to the assay mixture on the basis of hyaluronidase-activity in the unsupplemented control. Results All 16 S. uheris cultures produced free hyaluronidase as indicated by the development of clear zones surrounding streptococcal growth on hyaluronidase-test-agarplates. S. uheris strain CCM 6186 revealed highest hyaluronidase activity and was therefore selected for the isolation of the enzyme. Hyaluronidase could be isolated from the cell-free culture supernatant of this strain in a highly purified form by a series of purification steps (Table 1). Precipitation of hyaluronidase with ammonium sulfate at a final concentration of 60% increased the specific activity from 0.6 u/mg to 6.7 ulmg. Further purification was achieved by chromatography on DEAE-cellulose. Most of the enzyme was eluted from the adsorbent column with NaCI at concentrations between 0.07 and 0.1 molll (Fig. 1). Following gelfiltration on ultragel AC4 increased further the specific hyaluronidase activity to 390.5 ulmg (Fig. 2). Finally, isoelectric focussing (Fig. 3) yielded a homogeneous preparation of highly purified hyaluronidase with an isoelectric point of pH 4.9 and a molecular weight of 54000 D (Fig. 4). The purified enzyme exhibited maximal activity at pH 6.0 (Fig. 5) and 45°C (Fig. 6). The Michaelis constant was estimated to be 7.0 X 10-2 mg/ml (Fig. 7). Hyaluronidase activity increased in the presence of Ca++, Mg++, Mn++, Co++, Li+, and K+ for 40-50% and decreased with Zn++ and Cd++ for 30-40% at a final concentration of 10 mmolll respectively (Fig. 8).
Table 1. Purification of hyaluronidase from S. uberis, strain CCM 6186 Step of purification (u/mg)*
Specific activity factor
Purification
Culture supernatant (NH4hS04 precipitation DEAE-cellulose Gel-filtration Isoelectric focussing
0.6 6.7 196.4 390.5 1013.5
1 11.2 327.3 650.8 1689.2
* units hyaluronidase per mg protein
Hyaluronidase from Streptococcus uberis
-
49
E
::J
-:>.
>
u ttl
125
0.15
100 0.10
75
0
E
III
ttl "0
50
0.05
c:
0
to.
::J
25
U ttl Z
ttl
>-
X
5
10 Fractions
0.00
15
Fig. 1. Chromatography of hyaluronidase from S. uberis strain CCM 6186 on DEAEcellulose. Hyaluronidase was eluted with 10 mmolll phosphate buffer pH 6.8, containing NaCI at a linear gradient of 0.0 to 0.5 molll. NaCI (-), hyaluronidase-activity (A)
OD 280
E
0.15
::J
>
10 0.10
u
ttl
III
ttl "0
5
0.05
c:
oto.
::J
ttl :>. X
27
30
40
50
0.00
Fractions
Fig. 2. Gelfiltration of chromatographically purified hyaluronidase from S. uberis strain CCM 6186 on ultragel ACA44 • Hyaluronidase-activity (A), OD 280 (x- - - -x) 4 Zbl. Bakt. 27111
50
P. Schaufuss, R. Sting, W. Schaeg, and H. Blobel
~
l 3
,
E ::J
..
20
~
15
>>
to
QI 1/1
to
"0
10
c
~
::J
It
5.5 pH
5
iii
• 0.2
0.0
4.5
>-
:I:
6 7
5.0
0.1
18 20
25
30
Fractions
Fig. 3. Isoelectric focussing of hyaluronidase from S. uberis strain CCM 6186. Hyaluronidase activity (.A), OD Z80 (x- - - -x), pH (e) Fig. 4. Estimation of molecular weight of purified hyaluronidase from S. uberis strain CCM 6186 by SDS-PAGE. Bands 1, 2, 3, 4, 5, 6 and 7 represented uz-macroglobulin (180 kD), j3-gaiactosidase (116 kD), fructose-6-phosphate kinase (84 kD), pyruvate kinase (58 kD), fumarase (48 kD), lactate dehydrogenase (36.5 kD), and triosephosphate isomerase (26.6 kD). Approximately 2 Ilg of the purified hyaluronidase was loaded on the gel.
1.0 1.0
-. >-
>.-
>
>
uco QI
u
1\1
0.5
0.5
>
>
iii
iii
Ii ...
~
5.0
6.0
7.0
30
pH Fig. 5. Influence of pH on hyaluronidase activity (.A) Fig. 6. Influence of temperature on hyaluronidase activity (.A)
40
50
Temperature (·C )
60
Hyaluronidase from Streptococcus uberis
51
O.S
0.4 0.3 >-
0.2
.~
0.1
ti co CI> >
OO+-JLl-"...l-lL.I.--"'-..L.....JL.L....IL.L....f:';';-"':';' •
:; 0.1 CI>
.. 0.2 0.3 0.4 0.5
Fig. 7. Influence of Ca++, Mg++, Mn++, Co++, K+, Li+, Zn++ and Cd++ on hyaluronidase activity. Hyaluronidase activities with addition of cations were compared with hyaluronidase activity of the standard assay (0.0.)
1
V
60
40
20
-20
-10
10
20
30
40
1
[S] Fig. 8. Reciprocal (Lineweaver-Burk) plot of purified hyaluronidase from S. uberis strain CCM 6186. Using varying amounts of hyaluronic acid velocity of enzyme reaction (v) was expressed as mg hyaluronic acid split after incubation for 30 min at 37°C. [S] = mg hyaluronic acid/ml, hyaluronidase activity (.A)
52
P. Schaufuss, R. Sting, W. Schaeg, and H. Blobel Discussion
Hyaluronidase activities have been detected in culture media of streptococci belonging to serological groups A, B, C, G, H, L, in those of Streptococcus mitis (10) and s. salivarius (6). The enzyme, which was mainly produced during the logarithmic growth phase (4), depolymerized hyaluronic acid (14). Hyaluronidase, thus, could facilitate the dissemination of streptococci into mucous membranes or other tissues (5). It was, therefore, called "spreading factor" (3). Our studies revealed that S. uberis produced this enzyme in significant concentrations. Hyaluronidase could be purified to a homogeneous protein by precipitation with ammonium sulfate, chromatography on DEAE-cellulose, gelfiltration on AC~4 ultragel and isoelectric focussing. This was in agreement with the findings of Schaufup et al. (16) on hyaluronidase from S. equi and S. pyogenes (9). On the other hand, Abramson (1) reported that staphylococcal hyaluronidase consisted of multiple electrophoretic and chromatographic forms. The purified hyaluronidase from S. uberis had a molecular weight of approximately 54000 D as estimated by SDS-PAGE. A similar molecular weight had been determined for hyaluronidase from S. equi by Schaufup et al. (16) and for hyaluronidase from S. pyogenes (9). In contrast Gerlach and Kohler (10) found a molecular weight of 75000 D for hyaluronidase from S. pyogenes. These differences could possibly be explained by different methods of molecular weight determination. Purification of hyaluronidase by isoelectric focussing indicated an isoelectric point (PI) of pH 4.9. Schaufup et al. (16) reported an PI of pH 5 for hyaluronidase from S. equi. Smyth and Fehrenbach (18) described isoelectric points (PIs) of pH 4.4 and 4.3 for hyaluronidase preparations from streptococci of serological groups A and C respectively. Hallas and Widdowson (7) found PIs at pH 5.9, 4.5 and 4.6 for hyaluronidase from streptococci of serological groups A, C and G. In the present study a pH-optimum of 6 and a temperature-optimum of 45°C was found to be optimal for hyaluronidase activity of S. uberis. Similar datas were obtained for hyaluronidase of S. equi (16). The Michaelis constant (KM) of purified hyaluronidase from S. uberis was 7.0 x 10-2 mg/ml. Schaufup et al. (16) reported KM-values of 6.2 x 10-2 mg/ml for hyaluronidase of S. equi. Hill (9) described KM-values for S. pyogenes of 3.8 X 10-4 molll and Gerlach and Kohler (4) of 4.0 X 10-2 mg/ml. These present findings about hy:tluronidase production of S. uberis and the characterization of the purified enzyme could be of interest for further studies on pathogenicity of these streptococcal species. References 1. Abramson, c.: Staphylococcal hyaluronate lyase: Multiple electrophoretic and chromatographic forms. Arch. Biochem. Biophys. 121 (1967) 103-106 2. Benchetrit, L. c., S. L. Pahuja, E. D. Gray, and R. D. Edstrom: A sensitive method for the assay of hyaluronidase activity. Anal. Biochem. 79 (1977) 431-437 3. Duran-Reynals, F.: Tissue permeability and spreading factors in infection. Bact. Rev. 6 (1942) 197-252 4. Gerlach, D. and W. Kohler: Hyaluronate lyase from Streptococcus pyogenes. I. The production and isolation of hyaluronate lyase. Zbl. Bakt. Hyg., I. Abt. Orig. A 221 (1961) 333-339 5. Ginsburg, I.: Mechanism of cell and tissue injury induced by group A streptococci: Relation to poststreptococcal sequelae. J. Infect. Dis. 126 (1972) 294--340
Hyaluronidase from Streptococcus uberis
53
6. Hahn, G., W. Heeschen und H. Tolle: "Streptococcus". Eine Studie zur Struktur,
Biochemie, Kultur und Klassifizierung. Kiel. Milchw. Forschungsber. 22 (1970) 333-546 7. Hallas, G. and j. P. Widdowson: Antibody to hyaluronidase of streptococci of Lancefield groups C and G. In: S. E. Holm and P. Christensen (eds.), Basic Concepts of Streptococcal Diseases. Reedbooks Ltd., Chertsey, Surrey (1981) 8. Heukeshoven, J. and R. Dernick: Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 6 (1985) 103-112 9. Hill, j.: Purification and properties of streptococcal hyaluronate lyase. Infect. Immun. 14 (1976) 726-735 10. Kohler, W.: Die Serologie des Rheumatismus und der Streptokokkeninfektionen, 3. Auf!.]. A. Bart Verlag, Leipzig (1963) 11. Laemmli, W. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4 • Nature 227 (1970) 68~85 12. Lineweaver, H. and D. Burk: The determination of enzyme dissociation constants. ]. Am. Chern. Soc. 56 (1934) 658-666 13. Lowry, O. H., N.J. Rosebrough, A. 1. Farr, and R. J. Randall: Protein measurement with the Folin phenol reagent. J. BioI. Chern. 193 (1951) 265-275 14. Ludowieg, j., B. Vennesland, and A. Dorfman: The mechanism of action of hyaluronidases. ]. BioI. Chern. 236 (1961) 333-339 15. Schaeg, W., R. Bingol, and H. Blobel: Purification of penicillinase (~-lactamase) and acid phosphatase from Staphylococcus aureus in one procedure. Biochim. Biophys. Acta 268 (1972) 542-549 16. Schaufup, P., R. Sting, and H. Blobel: Purification and characterization of hyaluronidase from Streptococcus equi. X'h Lancefield International Symposium on Streptococci and Streptococcal Diseases, Cologne, FRG, 1.-4. Sept. 1987 17. Smith, R. F. and N. P. Willet: Rapid plate method for screening hyaluronidase and chondroitin sulfatase-producing microorganisms. Appl. Microbiol. 16 (1968) 1434-1436 18. Smyth, C. j. and F. J. Fehrenbach: Isoelectric analysis of haemolysins and enzymes from streptococci of groups A, C and G. Acta path. microbiol. scand. Sect. B 82 (1974) 860-870 19. Weber, K. and M. Osborn: The reliability of molecular weight determinations by dodecylsulfate-polyacrylamide gel electrophoresis. J. BioI. Chern. 244 (1969) 4406-4412 Dr. Peter Schaufup, Inst. f. Bakteriologie und Immunologie, Fachbereich Veterinarmedizin der Universitat Giel~en, Frankfurter Str. 107, D-6300 Giel~en