ANALYTICAL
BIOCHEMISTRY
A Rapid
65,
421-434 (1975)
Assay
Method
for
Hyaluronidase
C. J. COULSON AND R. GIRKIN' Pharmacological
Research Laboratories, Dagenham, Essex, RMlO
May & Baker 7X.9 U.K.
Received September 13. 1974; accepted November
Ltd.,
19. 1974
Hyaluronidase activity has been measured by a semimicro radiochemical method. 3H-Hyaluronic acid was prepared using hydrazine to remove some of the acetyl groups followed by reacetylation with tritiated acetic anhydride. Hydrolysis was carried out using an extract from rat liver lysosomes and a cruder extract from other tissues. At the completion of hydrolysis. cetylpyridinium chloride was added to precipitate unreacted substrate. Smaller molecular-weight oligosaccharides produced by hydrolysis remain in solution to be determined by liquid scintillation counting. This assay gives results that are similar to those obtained by the assay using Nacetylglucosamine end-group determination but, in the case of tissue extracts, in 1 hr instead of I8 hr.
In the course of work investigating the pathogenesis of various connective tissue diseases it became clear that there was no rapid assay for hyaluronidase. A semimicro radiochemical assay has been developed which is reproducible, rapid, and does not require large quantities of either substrate or enzyme. Hyaluronic acid obtained from human umbilical cord is treated with hydrazine in order to remove some of the acetyl groups located on glucosamine residues (1). These residues are replaced with 3H-labeled acetyl groups using tritiated acetic anhydride. The tritiated hyaluronic acid is purified using Sephadex G-100 and serves as a substrate for the enzyme reaction. The principle of the assay lies in the fact that cetylpyridinium chloride is able to precipitate hyaluronic acid but fails to precipitate smaller molecular-weight polysaccharides. The breakdown products of the reaction, therefore, remain in solution while the polymer is precipitated, and the soluble radioactivity can be estimated by liquid scintillation counting. The assay was developed using the enzyme activity from rat liver * lysosomes.
’ Present address: Huntingdon
Research Centre. Huntingdon, 427
Copyright @ 1975 by Academic Press, Inc. All tights of reproduction in any form reserved.
PEl8 6ES, U.K.
428
COULSON
AND
GIRKIN
MATERIALS
The following materials were obtained from Sigma (London) Ltd.: bovine testicular hyaluronidase (Type VI), hyaluronic acid sodium salt from umbilical cord, and blue dextran. Sephadex G-100 was from Pharmacia. British Drug Houses supplied cetyltrimethylammonium bromide and cetylpyridinium chloride which were both recrystallized before use. Tritiated acetic anhydride (500 mCi/mmole) was from the Radiochemical Centre, Amersham. All other materials used were of analytical reagent grade. The microtubes (volume 410 ~1) for enzyme assay were obtained from Hawksley (Lancing, Sussex, U.K.) and were centrifuged in a microfuge (Beckman 152A). METHODS 1. Preparation
of [3H]Hyaluronic
(a) Preparation
of partially
Acid deacylated
hyaluronic
acid. Five-tenths
gram hyaluronic acid and 5 ml hydrazine (anhydrous) were heated in a small carius tube (0.75 x 35 cm) at 98- 100°C for 18 hr. Hydrazine was removed by rotary evaporation at 55°C under vacuum. The dried material was dissolved in distilled water, dialyzed against distilled water, and filtered through Hyflo (Johns-Manville, London). Free amino groups were estimated by using the ninhydrin color reaction with glucosamine as a standard. The hyaluronic acid was dried and weighed, giving 0.35-0.4 g of 7-9% deacylated hyaluronic acid. (b) Reacyfation of partially deacylated hyaluronic acid. Fifty milligrams partially deacylated hyaluronic acid were dissolved in phosphate buffer 0.1 M. pH 10 (10 ml). The deacylated hyaluronic acid solution was placed in the 25-ml vessel attachment of a MSE top-drive homogenizer, together with 2 ml of a benzene solution of C3H]acetic anhydride (13.9 mg, 20 mCi). The homogenizer was driven for half an hour at a speed sufficient to maintain the water and benzene in an emulsified state. The material was kept at 12°C by immersing the homogenization vessel in a water bath. The mixture was allowed to stand for 1.5 hr at room temperature, was centrifuged, and the benzene removed by aspiration. (c) Purijcation of reacylated material. The hyaluronic acid solution (ca. 10 ml) was dialyzed against 5 liters of distilled water. Twenty to thirty drops of a saturated solution of cetyltrimethylammonium bromide were added until no further precipitation was observed (2). The precipitate was collected by centrifugation and redissolved in 2 M NaCI. The solution was dialyzed against 2 M NaCl (5 liters) for 6 hr. In order to
A
RAPID
ASSAY
FOR
HYALURONIDASE
429
remove cetyltrimethylammonium ions as the thiocyanate salt, 1 M potassium thiocyanate was added dropwise to the solution until no further precipitate was observed. The precipitate was removed by filtration through Hyflo. The filtrate containing the [3H]hyaluronic acid was dialyzed overnight against 5 liters distilled water and then for 6 hr against 5 liters 0.1 M acetate buffer, pH 3.6, containing 0.15 M NaCl, 0.001 M EDTA. The final volume of dialyzate was ca. 60 ml. A column (2 X 14 cm) of Sephadex G-100 Fine was prepared and washed with 1 liter of acetate buffer. Five milliliters of [3H]hyaluronic acid solution were loaded on the column and eluted with acetate buffer. Two-milliliter fractions were collected and the radioactivity in these fractions estimated by liquid scintillation counting. The elution pattern from the column is essentially the same as that noted for hyaluronic acid in Fig. 5. The [3H]hyaluronic acid was diluted with cold hyaluronic acid in 0.1 M formate buffer, pH 3.5, to give a concentration of 1.2 mglml. The specific activity of [3H]hyaluronic acid as freshly prepared was 0.413 &i/mg. The material was stored at -20°C. 2. Lysosomal
Preparation
A lysosomal fraction was prepared by a modification of the method of Dingle (3). The livers from 15 rats (Sprague-Dawley, 200-250 g) were homogenized (1: 3 by volume) in 0.25 M sucrose containing 1Oe3 M EDTA. The resulting homogenate was centrifuged at 1OOOgfor 15 min and the pellet discarded. The supernatant was centrifuged at 13 ,OOOg for 15 min. The resulting supernatant was retained while the pellet was resuspended in 5 ml of 0.25 M sucrose/l0-3 M EDTA, and spun at 13,000g for 5 min. The second supernatant was added to the first and centrifuged at 35,000g for 30 min. The pellet obtained was resuspended in 10 ml sucrose/EDTA containing 0.1 ml Triton X- 100. After 30-min incubation at 37°C the lysosomal fragments were removed by centrifugation at 35,000g for 25 min and the supernatant used for the subsequent studies. 3. Tissue Homogenization
The lungs and livers were taken from four Hooded Listar rats, and the livers and kidneys from four guinea-pigs. The organs were then homogenized separately in 2.5 vol of 0.1 M formate buffer, pH 3.5. Acidwashed sand was used to grind the lungs before homogenization. The homogenates were then centrifuged at 30,OOOg for 10 min, and the precipitate discarded. Aliquots of the supernatant (25 ~1) were assayed for hyaluronidase activity in a similar way to that used for the lysosomal preparation except that a I-hr period was used.
430
COULSON
AND
GIRKIN
4. Measurement of the Enzyme Activity Hyaluronic acid (60 ,ug in 50 ,ul of 0.1 M formate buffer pH 3.5 containing 0.15 M NaCl), was incubated in microtubes with 50 ~1 of enzyme solution for 20 min at 37°C. The reaction was terminated by neutralization with 50 ~1 of 0.25 M disodium hydrogen phosphate. Fifty microliters of cetylpyridinium chloride (1% solution in water) were then added in order to precipitate the unchanged substrate. The resultant mixture was incubated for a further 30 min at 37°C to ensure complete precipitation and centrifuged for 10 min in the microfuge. A 50-,~l aliquot of the supernatant was extracted for counting in 10 ml of a scintillant composed of toluene/Triton X- 100 2 : 1 by volume and containing 5 g of 2,5-diphenyloxazole per liter. Duplicate blank tubes were carried through the procedure except that the enzyme was added just before the addition of disodium hydrogen phosphate. Radioactivity present in the blanks after precipitation was subtracted from that obtained in the tubes containing enzymes to give the radioactivity solubilized by the action of the enzyme. Activities were corrected to dpm by the external standard method. Protein was estimated by the method of Lowry et al. (4) using bovine serum albumin as the standard. RESULTS
Figure la shows that the rate of reaction is linear with enzyme concentration. The time course of reaction is also linear for 20 min and then begins to decline (Fig. 2). The substrate concentration profile is plotted in Fig. 3 as a doublereciprocal plot after Lineweaver and Burk (5). At hyaluronate concentrations greater than 0.4 mg/ml substrate inhibition occurs. The addition of 0.075 M sodium chloride abolishes this substrate inhibition at least up
FIG. 1. (a). Lysosomal extract (O-l.7 mg protein) was incubated with 60 pg hyaluronic acid in 100 ~1 0.1 M formate buffer, pH 3.5, containing 0.075 M NaCl for 20 min at 37°C. (b). Bovine testicular hyaluronidase (O-20 Fg) was incubated with 60 Fg hyaluronic acid in 100 ~1 0.1 M acetate buffer. pH 5.0, containing 0.075 M NaCl for 1 hr at 37°C.
A RAPID
ASSAY
FOR HYALURONIDASE
431
FIG. 2. Hyaluronic acid (50 pg) was incubated at 37°C in 100 ~1 0.1 M buffer containing 0.075 M NaCl, for the various times stated, with: (A) Rat liver lysosomal extract ( 1.7 mg), formate buffer pH 3.5; (0) Bovine testicular hyaluronidase (50 fig), acetate buffer pH 5.0.
to the higher hyaluronate concentration used (0.6 mglml). Sodium chloride, however, inhibits the enzyme raising the apparent K, from 1.2 mg/ml to 5 mgfml. Rat liver lysosomal hy~uronidase has a pH optimum of 3.5 (Fig. 4). The activity dropped to negligible levels at pH 4.4 and above. In order to show that the hyaluronic acid had been degraded into smaller oligosaccharides, the normal reaction mixture was incubated for 2 hr, the reaction was then stopped by the addition of sodium phosphate and cetylpyridinium chloride, and an aliquot loaded on to a Sephadex G-100 column. The material was eluted with distilled water and the radioactivity of the fractions estimated by liquid scintillation counting to give the radioactivity profile shown in Fig. 5. It can be seen that the cetylpyridinium chloride removes all but a very small part of the hyaluronic acid and leaves the smaller molecular-weight fragments in solution. Thus, hy~uronic acid is indeed degraded into a group of molecules of greatly reduced size.
FIG 3. Sixty microgr~s of [~Hlhyaluronic acid were incubated with 1.7 mg lysosomal extract for 20 miu at pH 3.5 and 37°C in 0.1 M formate buffer containing 0.075 M NaCl (0) and in formate buffer without salt (A).
432
COULSON
AND
GIRKIN
FIG. 4. Hyaluronic acid (60 pg) was incubated with 1.7 mg lysosomal extract for 20 min at 37°C in 100 ~1 0.1 M buffer, formate at pH 3.0-4.0, and acetate pH 4.2-5.0.
This assay has also been used to measure the activity of purified hyaluronidase from bovine testes. The method was essentially the same as that used for the rat liver enzyme. The results are shown in Fig. lb. It can be seen that the rate of reaction is proportional to enzyme concentration over the range tested up to 100 pg/ml final concentration. Above this value a plateau is reached. The time-course (Fig. 2) for the bovine enzyme differs somewhat from that for the rat lysosomal enzyme, being linear for at least 50 min. Hyaluronidase activity was also detected in tissue extracts of guineapig liver and kidney, rat liver and lung, and in the edema produced by the intra-articular injection of turpentine in rabbits (Table 1). We could not detect hyaluronidase activity in rabbit articular cartilage.
FIG. 5. Five-tenths milliliter [“H]hyaluronic acid (60 pg) was incubated with 0.5 ml lysosomal extract (17 mg protein) for 2 hr. The reaction was stopped by the addition of 0.5 ml 0.25 M disodium hydrogen phosphate followed by 0.5 ml cetylpyridinium chloride (CPC, 1% solution in water). After centrifugation 1 ml supernatant was loaded on to a Sephadex G-100 column (35 X 1.5 cm) and eluted with distilled water at a flow rate of 6 ml/hr. Fractions of 1.5 ml were collected and 0.5-ml aliquots from each fraction were estimated for radioactivity by liquid scintillation counting. Omissions: None (-----); CPC (---); enzyme, CPC (-----); enzyme (---).
A RAPID
ASSAY
FOR
TABLE SPECIFIC
Species Rat Rat Guinea pig Guinea pig Rabbit Rabbit
ACTIVITIES
433
HYALURONIDASE
1
OF VARIOUS
TISSUES
AND SPECIE+
Mean specific activity (+SE) pg hyahtronate/mg protein/hour
Tissue Liver Lung Liver Kidney Synovial fluid from turpentine-induced edema Articular cartilage
8.40 13.47 16.49 23.38 11.39
a 0.69 5 4.14 i 2.08 r 2.48 -c 0.68 0
a Sixty microliters of hyaluronic acid were incubated with 25 ~1 tissue extract in 100 ~1 formate buffer 0.1 M, pH 3.5. containing 0.075 M NaCl at 37°C for 1 hr.
DISCUSSION
Aronson and Davidson (6) purified rat liver lysosomal hyaluronidase, 1300-fold, using as an assay the determination of end-group N-acetylglucosamine released during the reaction. The results of this new assay are in agreement with respect to the pH optimum and the time course of the reaction. In addition we obtain similar results with respect to the substrate concentration profile and the inhibition by high sodium chloride concentrations. However the apparent K, of 1.2 mg/ml for hyaluronic acid which we obtain does not agree with the figure of 0.08 mg/ml obtained by Aronson and Davidson (7). This discrepancy could arise from hyaluronic acid binding to a component of the crude extract. The two assays are in broad agreement, therefore, although one was carried out on a crude lysosomal extract and the other on a highly purified enzyme preparation. This assay can also be used to detect the presence of hyaluronidase activity in crude tissue extracts in a time for assay (1 hr) which is much shorter than the incubation period of 18 hr required by Bollet, Bonner, and Nance (8) with the N-acetylglucosamine end-group assay. We were not able to detect enzyme activity in rabbit articular cartilage, in agreement with Bollet (9). ACKNOWLEDGMENTS We are indebted to Dr. R. F. Collins for his helpful advice and to Mr. V. J. Smith and Mr. P. S. Thorne for their excellent technical assistance.
REFERENCES 1. Matsushima, Y.. and Fujii, N. (1957) BUM Chem. 2. Korn, E. D. (1959) J. Biol. Chem. 234, 1375.
Sot.
Jap.
30, 48.
434
COULSON
AND
GIRKIN
3. Dingle, J. T. (1961) Biochem. J. 79, 509. 4. Lowry, 0. H., Rosebrough, N. J., Farr. A. L., and Randall, R. J. (1951) J. Biol. Clzem. 193, 365.
5. 6. 7. 8.
Lineweaver, H., and Burk, D. (1934) J. Amer. Chem. Sot. 56, 658. Aronson, N. N., and Davidson. E. A. (1967a) .I. Biol. Chem. 242, 437. Aronson, N. N., and Davidson, E. A. (1967b) J. Biol. Chem. 242, 441. Ballet, A. J., Bonner, W. M.. and Name, J. L. (1963) J. Biol. Chen. 9. Ballet, A. J. (1967) Advnn. Interrz. Med. 13, 33.
238,
3522.
BIOCHEMISTRY
A Rapid
65,
421-434 (1975)
Assay
Method
for
Hyaluronidase
C. J. COULSON AND R. GIRKIN' Pharmacological
Research Laboratories, Dagenham, Essex, RMlO
May & Baker 7X.9 U.K.
Received September 13. 1974; accepted November
Ltd.,
19. 1974
Hyaluronidase activity has been measured by a semimicro radiochemical method. 3H-Hyaluronic acid was prepared using hydrazine to remove some of the acetyl groups followed by reacetylation with tritiated acetic anhydride. Hydrolysis was carried out using an extract from rat liver lysosomes and a cruder extract from other tissues. At the completion of hydrolysis. cetylpyridinium chloride was added to precipitate unreacted substrate. Smaller molecular-weight oligosaccharides produced by hydrolysis remain in solution to be determined by liquid scintillation counting. This assay gives results that are similar to those obtained by the assay using Nacetylglucosamine end-group determination but, in the case of tissue extracts, in 1 hr instead of I8 hr.
In the course of work investigating the pathogenesis of various connective tissue diseases it became clear that there was no rapid assay for hyaluronidase. A semimicro radiochemical assay has been developed which is reproducible, rapid, and does not require large quantities of either substrate or enzyme. Hyaluronic acid obtained from human umbilical cord is treated with hydrazine in order to remove some of the acetyl groups located on glucosamine residues (1). These residues are replaced with 3H-labeled acetyl groups using tritiated acetic anhydride. The tritiated hyaluronic acid is purified using Sephadex G-100 and serves as a substrate for the enzyme reaction. The principle of the assay lies in the fact that cetylpyridinium chloride is able to precipitate hyaluronic acid but fails to precipitate smaller molecular-weight polysaccharides. The breakdown products of the reaction, therefore, remain in solution while the polymer is precipitated, and the soluble radioactivity can be estimated by liquid scintillation counting. The assay was developed using the enzyme activity from rat liver * lysosomes.
’ Present address: Huntingdon
Research Centre. Huntingdon, 427
Copyright @ 1975 by Academic Press, Inc. All tights of reproduction in any form reserved.
PEl8 6ES, U.K.
428
COULSON
AND
GIRKIN
MATERIALS
The following materials were obtained from Sigma (London) Ltd.: bovine testicular hyaluronidase (Type VI), hyaluronic acid sodium salt from umbilical cord, and blue dextran. Sephadex G-100 was from Pharmacia. British Drug Houses supplied cetyltrimethylammonium bromide and cetylpyridinium chloride which were both recrystallized before use. Tritiated acetic anhydride (500 mCi/mmole) was from the Radiochemical Centre, Amersham. All other materials used were of analytical reagent grade. The microtubes (volume 410 ~1) for enzyme assay were obtained from Hawksley (Lancing, Sussex, U.K.) and were centrifuged in a microfuge (Beckman 152A). METHODS 1. Preparation
of [3H]Hyaluronic
(a) Preparation
of partially
Acid deacylated
hyaluronic
acid. Five-tenths
gram hyaluronic acid and 5 ml hydrazine (anhydrous) were heated in a small carius tube (0.75 x 35 cm) at 98- 100°C for 18 hr. Hydrazine was removed by rotary evaporation at 55°C under vacuum. The dried material was dissolved in distilled water, dialyzed against distilled water, and filtered through Hyflo (Johns-Manville, London). Free amino groups were estimated by using the ninhydrin color reaction with glucosamine as a standard. The hyaluronic acid was dried and weighed, giving 0.35-0.4 g of 7-9% deacylated hyaluronic acid. (b) Reacyfation of partially deacylated hyaluronic acid. Fifty milligrams partially deacylated hyaluronic acid were dissolved in phosphate buffer 0.1 M. pH 10 (10 ml). The deacylated hyaluronic acid solution was placed in the 25-ml vessel attachment of a MSE top-drive homogenizer, together with 2 ml of a benzene solution of C3H]acetic anhydride (13.9 mg, 20 mCi). The homogenizer was driven for half an hour at a speed sufficient to maintain the water and benzene in an emulsified state. The material was kept at 12°C by immersing the homogenization vessel in a water bath. The mixture was allowed to stand for 1.5 hr at room temperature, was centrifuged, and the benzene removed by aspiration. (c) Purijcation of reacylated material. The hyaluronic acid solution (ca. 10 ml) was dialyzed against 5 liters of distilled water. Twenty to thirty drops of a saturated solution of cetyltrimethylammonium bromide were added until no further precipitation was observed (2). The precipitate was collected by centrifugation and redissolved in 2 M NaCI. The solution was dialyzed against 2 M NaCl (5 liters) for 6 hr. In order to
A
RAPID
ASSAY
FOR
HYALURONIDASE
429
remove cetyltrimethylammonium ions as the thiocyanate salt, 1 M potassium thiocyanate was added dropwise to the solution until no further precipitate was observed. The precipitate was removed by filtration through Hyflo. The filtrate containing the [3H]hyaluronic acid was dialyzed overnight against 5 liters distilled water and then for 6 hr against 5 liters 0.1 M acetate buffer, pH 3.6, containing 0.15 M NaCl, 0.001 M EDTA. The final volume of dialyzate was ca. 60 ml. A column (2 X 14 cm) of Sephadex G-100 Fine was prepared and washed with 1 liter of acetate buffer. Five milliliters of [3H]hyaluronic acid solution were loaded on the column and eluted with acetate buffer. Two-milliliter fractions were collected and the radioactivity in these fractions estimated by liquid scintillation counting. The elution pattern from the column is essentially the same as that noted for hyaluronic acid in Fig. 5. The [3H]hyaluronic acid was diluted with cold hyaluronic acid in 0.1 M formate buffer, pH 3.5, to give a concentration of 1.2 mglml. The specific activity of [3H]hyaluronic acid as freshly prepared was 0.413 &i/mg. The material was stored at -20°C. 2. Lysosomal
Preparation
A lysosomal fraction was prepared by a modification of the method of Dingle (3). The livers from 15 rats (Sprague-Dawley, 200-250 g) were homogenized (1: 3 by volume) in 0.25 M sucrose containing 1Oe3 M EDTA. The resulting homogenate was centrifuged at 1OOOgfor 15 min and the pellet discarded. The supernatant was centrifuged at 13 ,OOOg for 15 min. The resulting supernatant was retained while the pellet was resuspended in 5 ml of 0.25 M sucrose/l0-3 M EDTA, and spun at 13,000g for 5 min. The second supernatant was added to the first and centrifuged at 35,000g for 30 min. The pellet obtained was resuspended in 10 ml sucrose/EDTA containing 0.1 ml Triton X- 100. After 30-min incubation at 37°C the lysosomal fragments were removed by centrifugation at 35,000g for 25 min and the supernatant used for the subsequent studies. 3. Tissue Homogenization
The lungs and livers were taken from four Hooded Listar rats, and the livers and kidneys from four guinea-pigs. The organs were then homogenized separately in 2.5 vol of 0.1 M formate buffer, pH 3.5. Acidwashed sand was used to grind the lungs before homogenization. The homogenates were then centrifuged at 30,OOOg for 10 min, and the precipitate discarded. Aliquots of the supernatant (25 ~1) were assayed for hyaluronidase activity in a similar way to that used for the lysosomal preparation except that a I-hr period was used.
430
COULSON
AND
GIRKIN
4. Measurement of the Enzyme Activity Hyaluronic acid (60 ,ug in 50 ,ul of 0.1 M formate buffer pH 3.5 containing 0.15 M NaCl), was incubated in microtubes with 50 ~1 of enzyme solution for 20 min at 37°C. The reaction was terminated by neutralization with 50 ~1 of 0.25 M disodium hydrogen phosphate. Fifty microliters of cetylpyridinium chloride (1% solution in water) were then added in order to precipitate the unchanged substrate. The resultant mixture was incubated for a further 30 min at 37°C to ensure complete precipitation and centrifuged for 10 min in the microfuge. A 50-,~l aliquot of the supernatant was extracted for counting in 10 ml of a scintillant composed of toluene/Triton X- 100 2 : 1 by volume and containing 5 g of 2,5-diphenyloxazole per liter. Duplicate blank tubes were carried through the procedure except that the enzyme was added just before the addition of disodium hydrogen phosphate. Radioactivity present in the blanks after precipitation was subtracted from that obtained in the tubes containing enzymes to give the radioactivity solubilized by the action of the enzyme. Activities were corrected to dpm by the external standard method. Protein was estimated by the method of Lowry et al. (4) using bovine serum albumin as the standard. RESULTS
Figure la shows that the rate of reaction is linear with enzyme concentration. The time course of reaction is also linear for 20 min and then begins to decline (Fig. 2). The substrate concentration profile is plotted in Fig. 3 as a doublereciprocal plot after Lineweaver and Burk (5). At hyaluronate concentrations greater than 0.4 mg/ml substrate inhibition occurs. The addition of 0.075 M sodium chloride abolishes this substrate inhibition at least up
FIG. 1. (a). Lysosomal extract (O-l.7 mg protein) was incubated with 60 pg hyaluronic acid in 100 ~1 0.1 M formate buffer, pH 3.5, containing 0.075 M NaCl for 20 min at 37°C. (b). Bovine testicular hyaluronidase (O-20 Fg) was incubated with 60 Fg hyaluronic acid in 100 ~1 0.1 M acetate buffer. pH 5.0, containing 0.075 M NaCl for 1 hr at 37°C.
A RAPID
ASSAY
FOR HYALURONIDASE
431
FIG. 2. Hyaluronic acid (50 pg) was incubated at 37°C in 100 ~1 0.1 M buffer containing 0.075 M NaCl, for the various times stated, with: (A) Rat liver lysosomal extract ( 1.7 mg), formate buffer pH 3.5; (0) Bovine testicular hyaluronidase (50 fig), acetate buffer pH 5.0.
to the higher hyaluronate concentration used (0.6 mglml). Sodium chloride, however, inhibits the enzyme raising the apparent K, from 1.2 mg/ml to 5 mgfml. Rat liver lysosomal hy~uronidase has a pH optimum of 3.5 (Fig. 4). The activity dropped to negligible levels at pH 4.4 and above. In order to show that the hyaluronic acid had been degraded into smaller oligosaccharides, the normal reaction mixture was incubated for 2 hr, the reaction was then stopped by the addition of sodium phosphate and cetylpyridinium chloride, and an aliquot loaded on to a Sephadex G-100 column. The material was eluted with distilled water and the radioactivity of the fractions estimated by liquid scintillation counting to give the radioactivity profile shown in Fig. 5. It can be seen that the cetylpyridinium chloride removes all but a very small part of the hyaluronic acid and leaves the smaller molecular-weight fragments in solution. Thus, hy~uronic acid is indeed degraded into a group of molecules of greatly reduced size.
FIG 3. Sixty microgr~s of [~Hlhyaluronic acid were incubated with 1.7 mg lysosomal extract for 20 miu at pH 3.5 and 37°C in 0.1 M formate buffer containing 0.075 M NaCl (0) and in formate buffer without salt (A).
432
COULSON
AND
GIRKIN
FIG. 4. Hyaluronic acid (60 pg) was incubated with 1.7 mg lysosomal extract for 20 min at 37°C in 100 ~1 0.1 M buffer, formate at pH 3.0-4.0, and acetate pH 4.2-5.0.
This assay has also been used to measure the activity of purified hyaluronidase from bovine testes. The method was essentially the same as that used for the rat liver enzyme. The results are shown in Fig. lb. It can be seen that the rate of reaction is proportional to enzyme concentration over the range tested up to 100 pg/ml final concentration. Above this value a plateau is reached. The time-course (Fig. 2) for the bovine enzyme differs somewhat from that for the rat lysosomal enzyme, being linear for at least 50 min. Hyaluronidase activity was also detected in tissue extracts of guineapig liver and kidney, rat liver and lung, and in the edema produced by the intra-articular injection of turpentine in rabbits (Table 1). We could not detect hyaluronidase activity in rabbit articular cartilage.
FIG. 5. Five-tenths milliliter [“H]hyaluronic acid (60 pg) was incubated with 0.5 ml lysosomal extract (17 mg protein) for 2 hr. The reaction was stopped by the addition of 0.5 ml 0.25 M disodium hydrogen phosphate followed by 0.5 ml cetylpyridinium chloride (CPC, 1% solution in water). After centrifugation 1 ml supernatant was loaded on to a Sephadex G-100 column (35 X 1.5 cm) and eluted with distilled water at a flow rate of 6 ml/hr. Fractions of 1.5 ml were collected and 0.5-ml aliquots from each fraction were estimated for radioactivity by liquid scintillation counting. Omissions: None (-----); CPC (---); enzyme, CPC (-----); enzyme (---).
A RAPID
ASSAY
FOR
TABLE SPECIFIC
Species Rat Rat Guinea pig Guinea pig Rabbit Rabbit
ACTIVITIES
433
HYALURONIDASE
1
OF VARIOUS
TISSUES
AND SPECIE+
Mean specific activity (+SE) pg hyahtronate/mg protein/hour
Tissue Liver Lung Liver Kidney Synovial fluid from turpentine-induced edema Articular cartilage
8.40 13.47 16.49 23.38 11.39
a 0.69 5 4.14 i 2.08 r 2.48 -c 0.68 0
a Sixty microliters of hyaluronic acid were incubated with 25 ~1 tissue extract in 100 ~1 formate buffer 0.1 M, pH 3.5. containing 0.075 M NaCl at 37°C for 1 hr.
DISCUSSION
Aronson and Davidson (6) purified rat liver lysosomal hyaluronidase, 1300-fold, using as an assay the determination of end-group N-acetylglucosamine released during the reaction. The results of this new assay are in agreement with respect to the pH optimum and the time course of the reaction. In addition we obtain similar results with respect to the substrate concentration profile and the inhibition by high sodium chloride concentrations. However the apparent K, of 1.2 mg/ml for hyaluronic acid which we obtain does not agree with the figure of 0.08 mg/ml obtained by Aronson and Davidson (7). This discrepancy could arise from hyaluronic acid binding to a component of the crude extract. The two assays are in broad agreement, therefore, although one was carried out on a crude lysosomal extract and the other on a highly purified enzyme preparation. This assay can also be used to detect the presence of hyaluronidase activity in crude tissue extracts in a time for assay (1 hr) which is much shorter than the incubation period of 18 hr required by Bollet, Bonner, and Nance (8) with the N-acetylglucosamine end-group assay. We were not able to detect enzyme activity in rabbit articular cartilage, in agreement with Bollet (9). ACKNOWLEDGMENTS We are indebted to Dr. R. F. Collins for his helpful advice and to Mr. V. J. Smith and Mr. P. S. Thorne for their excellent technical assistance.
REFERENCES 1. Matsushima, Y.. and Fujii, N. (1957) BUM Chem. 2. Korn, E. D. (1959) J. Biol. Chem. 234, 1375.
Sot.
Jap.
30, 48.
434
COULSON
AND
GIRKIN
3. Dingle, J. T. (1961) Biochem. J. 79, 509. 4. Lowry, 0. H., Rosebrough, N. J., Farr. A. L., and Randall, R. J. (1951) J. Biol. Clzem. 193, 365.
5. 6. 7. 8.
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