Characterization of Hyaluronidase Isolated from Agkistrodon contortrix contortrix (Southern Copperhead) Venom

Archives of Biochemistry and Biophysics Vol. 386, No. 2, February 15, pp. 154 –162, 2001 doi:10.1006/abbi.2000.2204, available online at http://www.idealibrary.com on

Characterization of Hyaluronidase Isolated from Agkistrodon contortrix contortrix (Southern Copperhead) Venom 1 Kenzo Kudo and Anthony T. Tu 2 Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523

Received May 16, 2000, and in revised form October 24, 2000; published online January 19, 2001

Snake venoms are a rich source of enzymes including many hydrolytic enzymes. Some enzymes such as phospholipase A 2 , proteolytic enzymes, and phosphodiesterases are well characterized. However many enzymes, such as the glycosidase, hyaluronidase, have not been studied extensively. Here we describe the characterization of snake venom hyaluronidase. In order to determine which venom was the best source for isolation of the enzyme, the hyaluronidase activity of 19 venoms from Elapidae, Viperidae, and Crotalidae snakes was determined. Since Agkistrodon contortrix contortrix venom showed the highest activity, this venom was used for purification of hyaluronidase. Molecular weight was determined by matrix-assisted laser desorption ionization mass spectroscopy and was found to be 59,290 Da. The molecular weight value as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 61,000 Da. Substrate specificity studies indicated that the snake venom enzyme was specific only for hyaluronan and did not hydrolyze similar polysaccharides of chondroitin, chondroitin sulfate A (chondroitin 4-sulfate), chondroitin sulfate B (dermatan sulfate), chondroitin sulfate C (chondroitin 6-sulfate), chondroitin sulfate D, chondroitin sulfate E, or heparin. The enzyme is an endo-glycosidase without exo-glycosidase activity, as it did not hydrolyze p-nitrophenyl-␤-D-glucuronide or p-nitrophenyl-N-acetyl-␤-D-glucosaminide. The main hydrolysis products from hyaluronan were hexa- and tetrasaccharides with N-acetylglucosamine at the reducing terminal. The cleavage point is at the ␤1,4glycosidic linkage and not at the ␤1,3-glycosidic linkage. Thus, snake venom hyaluronidase is an 1

This work is supported by the Tu Family Foundation. To whom correspondence should be addressed. Fax: (970) 4916313. E-mail: [email protected]. 2

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endo-␤-N-acetylhexosaminidase specific for hyaluronan. © 2001 Academic Press Key Words: snake venom; glycosidase; hyaluronidase; endo-␤-N-acetylhexosaminidase; hyaluronoglucosaminidase.

Snake venom contains a wide variety of compounds including toxic, nontoxic, enzymatic and nonenzymatic proteins (1–3). Many of the toxic proteins, especially neurotoxins and myotoxins, have been well studied (4 – 6). Snake venom is a rich source of enzymes including a variety both of hydrolytic and of a few nonhydrolytic enzymes. Some enzymes are especially well characterized; for example, phospholipase A 2, phosphodiesterase, and proteolytic enzymes, including fibrinolytic enzymes, have been isolated from various snake venoms and studied extensively (7–10). Hyaluronidase is an important enzyme found in many sources including tear, sperma, placenta, uterus, liver, kidney, eye, skin, spleen, and testes, and also in leeches (11–14). The enzyme is produced by a number of bacteria and some mammalian tumors, including prostate carcinoma and human carcinoma cell lines derived from lung and cervix tumors (15–18). Hyaluronidase is also commonly found in various animal venoms because of its action as a spreading factor (19 –23). Hyaluronan is considered to be a cementing substance joining different cells in the tissues. Venom hyaluronidase hydrolyzes hyaluronan to facilitate the spread of toxins and venoms (24, 25). Occurrence of hyaluronidase in snake venoms is very common; however, very few biochemical characterizations have been made despite its important biological action. To better understand this important enzyme, hyaluronidase was isolated and characterized. In order to determine which snake venom contained the highest 0003-9861/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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enzyme activity, the activity of 19 venoms was investigated prior to isolation. Among them the venom of Agkistrodon contortrix contortrix (southern copperhead) showed the highest activity. Therefore, hyaluronidase was isolated from this venom. The biochemical characterization of hyaluronidase was made and the results are presented in this report. MATERIALS AND METHODS

Materials Lyophilized crude venom from A. contortrix contortrix was purchased from Miami Serpentarium Laboratories (Punta Gorda, FL). Sephacryl S-200 HR and low molecular weight calibration kit were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). CM-Sephadex C-25, hexadecyltrimethylammonium bromide, porcine intestinal mucosa chondroitin sulfate B, heparin, p-nitrophenyl-␤-Dglucuronide, and ␤-glucuronidase from Escherichia coli were from Sigma Chemical Co. (St. Louis, MO). Bovine testes hyaluronidase was from Calbiochem (San Diego, CA). Human umbilical cord hyaluronan, chondroitin, sturgeon notochord chondroitin sulfate A, shark cartilage chondroitin sulfate C, shark cartilage chondroitin sulfate D, squid cartilage chondroitin sulfate E, p-nitrophenyl-Nacetyl-␤-D-glucosaminide, and ␤-N-acetylhexosaminidase from jack beans were from Seikagaku America Inc. (Falmouth, MA). All other chemicals and reagents were of the first grade commercially available.

Methods Hyaluronidase assay. The hyaluronidase activity was determined turbidimetrically by the method of Ferrante (26). Aliquots of 50 ␮l of the enzyme solution were added to 70 ␮g of hyaluronan in 0.45 ml of 0.2 M sodium acetate buffer, pH 6.0, containing 0.15 M NaCl. The mixture was incubated at 37°C for 15 min, and then the reactions were terminated by the addition of 1 ml of 2.5% hexadecyltrimethylammonium bromide in 2% NaOH solution. After 5 min, the optical density of each sample was read by spectrophotometer at 400 nm and then blank-corrected. All assays were performed in duplicate. Bovine testes hyaluronidase was also used for comparison. One unit is defined as the amount of enzyme that will cause the same turbidity reduction as 1.0 unit of international standard preparation. Substrate specificity of the hyaluronidase. Potential substrates chondroitin, chondroitin sulfate A (chondroitin 4-sulfate), chondroitin sulfate B (dermatan sulfate), chondroitin sulfate C (chondroitin 6-sulfate), chondroitin sulfate D, chondroitin sulfate E, and heparin were used to determine the substrate specificity of the purified enzyme. The assays were set up with 50 ␮g of various substrates and 100 units of the enzyme preparation in a total volume of 500 ␮l of 0.2 M sodium acetate buffer, pH 6.0, containing 0.15 M NaCl. The reaction mixtures were incubated at 37°C for 1 h and the degradation of hyaluronan and other substrates was determined by monitoring the decrease in optical density at 400 nm. When the enzyme activity was measured using chondroitin, the Morgan-Elson method (27) was used instead of the turbidimetric method, because turbidity was not produced by adding hexadecyltrimethylammonium bromide. ␤-glucuronidase and ␤-N-acetylhexosaminidase activities of snake hyaluronidase were measured by the method of Barrett using p-nitrophenyl-␤-D-glucuronide and p-nitrophenyl-N-acetyl-␤D-glucosaminide (28). Isolation of hyaluronidase from crude venom. All purification steps were carried out at 4°C. Five hundred milligrams of A. contortrix contortrix venom was dissolved in 5 ml of 0.02 M sodium phosphate buffer, pH 6.0, containing 0.15 M NaCl. The dissolved venom was centrifuged at 10,000g for 20 min to remove insoluble material.

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The supernatant liquid was applied to a Sephacryl S-200 HR column (2.6 ⫻ 50 cm) equilibrated with the same buffer. The proteins were eluted with the buffer at a flow rate of 12 ml/h, and 3-ml fractions were collected. All of the fractions were assayed for hyaluronidase activity as described by Ferrante (26), and the protein content of each fraction was monitored at 280 nm by a spectrophotometer. The fractions containing high hyaluronidase activity were pooled and loaded on a CM-Sephadex C-25 column (1.5 ⫻ 10 cm), which was equilibrated with 0.02 M sodium phosphate buffer, pH 6.0, containing 0.15 M NaCl. The column was washed with the same buffer followed by fractionation using a linear gradient of 0.15 to 0.8 M NaCl in 160 ml of 0.02 M sodium phosphate buffer, pH 5.5, at a flow rate of 10 ml/h, and collection of 2.5 ml fractions. Finally, the fractions containing the enzyme activity were pooled and concentrated using a Centriplus ultrafiltration device. Polyacrylamide gel electrophoresis (PAGE). 3 Sodium dodecyl sulfate (SDS)-PAGE was performed on a PhastSystem electrophoresis module (Amersham Pharmacia Biotech) according to the instructions in the manual. For SDS-PAGE, PhastGel Gradient 10 –15% gels were used. The samples were incubated in 2.5% SDS and 5% 2-mercaptoethanol at 100°C for 5 min. After electrophoresis, gels were stained with 0.25% Coomassie brilliant blue R-250 and destained with 25% ethanol-8% acetic acid. Mass spectrometry. The mass was determined by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry in the positive ion mode. ␣-Cyano-4-hydroxycinnamic acid was used as MALDI matrix. Isoelectric focusing. For isoelectric focusing, PhastGel IEF 3-9 was used. After focusing, the proteins on the gels were fixed with a 20% trichloroacetic acid solution and stained with PhastGel Blue R solution. The gels were destained with 25% ethanol-8% acetic acid for protein band observation. Protein assay. Protein concentrations were determined by the Bradford method (29) using the Pierce Coomassie protein assay reagent. Bovine serum albumin (BSA) was used as a protein standard. Oligosaccharide analysis. Uronic acid concentration was determined by the carbazole method of Bitter and Muir (30), using glucuronic acid as the standard. Reducing N-acetylglucosamine units were determined by the method of Reissig et al. (27), which is a modification of the Morgan-Elson reaction. N-Acetyl-D-glucosamine was used as the standard for the reduced terminal unit determination. Enzyme digestion and thin-layer chromatography (TLC). Hydrolysis of hyaluronan was performed using 700 units of purified hyaluronidase and 200 ␮g of hyaluronan in a total volume of 100 ␮l of 0.2 M sodium acetate buffer, pH 6.0, containing 0.15 M NaCl and 0.05% BSA at 37°C. After incubations of 8, 24, and 48 h, reactions were terminated by placing the samples in a boiling water bath at 100°C for 2 min. The cooled digestion mixture was centrifuged at 10,000g for 20 min to remove the precipitated protein. Digestion with 700 units of bovine testes hyaluronidase was performed in the same manner, except that the buffer did not contain BSA. For the hydrolysis of tetrasaccharide with ␤-glucuronidase, 20 ␮g of substrate and 200 Fishman units of enzyme in a total volume of 80 ␮l of 0.06 M sodium potassium phosphate buffer, pH 6.8, were incubated at 37°C for 3 h. For ␤-N-acetylhexosaminidase digestion of the tetrasaccharide, the substrate (20 ␮g) was mixed with 1 unit of enzyme in a total volume of 80 ␮l of 0.15 M citrate phosphate buffer,

3 Abbreviations used: BSA, bovine serum albumin; GTA buffer, 3,3-dimethylglutaric acid/tris(hydroxymethyl)aminomethane/2-amino-2-methyl-1,3-propanediol buffer; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight.

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KUDO AND TU TABLE I

Hyaluronidase Activity of Various Snake Venoms

Family name

Genus and species

Common name

Specific activity a (unit/mg)

Elapidae Bungarus fasciatus Naja naja Naja naja atra Ophiophagus hannah

Banded krait Indian cobra Formosan cobra King cobra

4.3 3.3 89.1 18.8

Bitis gabonica Vipera russelli (Thailand)

Gaboon viper Russell’s viper

170.8 108.5

Agkistrodon bilineatus Agkistrodon contortrix contortrix Agkistrodon contortrix laticinctus Agkistrodon halys blomhoffii Agkistrodon piscivorus leucostoma Agkistrodon piscivorus piscivorus Agkistrodon rhodostoma Bothrops atrox Crotalus adamanteus Crotalus atrox Crotalus basiliscus Crotalus horridus horridus Trimeresurus flavoviridis

Tropical moccasin Southern copperhead Broadbanded copperhead Japanese mamushi Western cottonmouth moccasin Eastern cottonmouth moccasin Malayan pit viper

60.3 243.0 118.4 72.0 207.3 109.3 92.0 73.1 67.2 148.7 207.7 156.8 41.6

Viperidae

Crotalidae

a

Eastern diamondback rattlesnake Western diamondback rattlesnake Mexican west-coast rattlesnake Timber rattlesnake Japanese habu

Specific activity: unit per mg of lyophilized venom.

pH 5.0, and incubated at 37°C for 3 h. The reactions were terminated by heating at 100°C for 2 min. Digestion products of hyaluronan were separated by thin-layer chromatography using a silica gel (31). Isopropanol:water (66:34) containing 0.05 M NaCl was used for the development in TLC. The spots containing oligosaccharides were visualized with the orcinolH 2SO 4-staining method. Other biochemical characterizations. For determination of the optimum pH for enzyme activity, 0.15 M GTA buffer containing 0.15 M NaCl was used. GTA buffer is a mixture of three compounds: 3,3-dimethylglutaric acid, tris(hydroxymethyl)aminomethane, and 2-amino-2-methyl-1,3-propanediol. The GTA solution has buffer capacity from 3.5 to 10.0. For the optimum temperature study, the enzyme activity at various temperatures was measured under the standard assay conditions. For the optimum NaCl concentration study, 0.1 M sodium acetate buffer, pH 6.0, containing various NaCl concentrations was used.

RESULTS

Purification of Hyaluronidase Hyaluronidase is a common enzyme in snake venoms; however, the enzyme has not been isolated or studied extensively. Before isolating the enzyme, a variety of snake venoms was examined for hyaluronidase activity. Altogether 19 venoms of snakes from Elapidae, Viperidae, and Crotalidae were investigated (Table I). All venom investigated showed hyaluronidase activity. In general, Crotalidae and Viperidae venoms showed higher hyaluronidase activity than Elapidae venoms. Specifically, the venom of A. contortrix contor-

trix (southern copperhead) showed the highest specific activity. Because of its high activity and its availability, the venom of A. contortrix contortrix was used for hyaluronidase purification. Purification of hyaluronidase was done in two steps using Sephacryl S-200 HR molecular sieve chromatography and CM-Sephadex C-25 ion-exchange chromatography (Fig. 1). Even though A. contortrix contortrix venom showed the highest activity among all venoms investigated, the concentration of hyaluronidase in the venom is very small. From 500 mg crude venom only 0.3 mg of purified hyaluronidase could be obtained (Table II). The recovery of the activity was 17% and the specific activity was increased by 277-fold as compared to the original crude venom. The progress of purification can be seen in the SDS gel (Fig. 2). In lane 2 of Fig. 2, 11 bands can be seen for crude venom. Six bands can be seen for the partially purified hyaluronidase (shaded area in Fig. 1A). The final purified fraction (shaded area in Fig. 1B) shows only one band (Fig. 2, lane 4). Electrophoresis in the presence of SDS and ␤-mercaptoethanol produced one major band corresponding to a subunit with an apparent molecular weight of 61,000 Da. The molecular weight of 61,000 Da was also obtained under nondissociating conditions (data not shown), indicating that hyaluronidase from A. contor-

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pendent methods: SDS-PAGE and MALDI-TOF mass spectrometry. By SDS-PAGE, the molecular weight of the hyaluronidase was estimated to be 61,000 Da, as discussed above. The molecular weight of purified hyaluronidase as determined by MALDI-TOF mass spectrometry is 59,290 Da. The relatively sharp peak in the mass spectrum also indicates the homogeneity of the purified hyaluronidase (Fig. 3). The smaller peak at a molecular weight of 29,623 Da was apparently derived from [M] 2⫹ in the mass spectrum. These independent methods gave comparable values for purified hyaluronidase. The isoelectric point of hyaluronidase was determined to be 9.0 by isoelectric focusing on the PhastSystem. This indicates that the hyaluronidase is a basic protein. Hydrolysis of Hyaluronan

FIG. 1. Purification of A. contortrix contortrix venom hyaluronidase. (A) Elution profile from the Sephacryl S-200 HR column. The protein concentration was determined by the absorbance at 280 nm (F) and hyaluronidase activity (E) of each fraction was assayed. The protein concentration was determined by dilution with buffer for proper reading. The fractions containing hyaluronidase [tubes 36 to 39 (shaded area)] were collected and applied to a CM-Sephadex C-25 column for further purification. (B) Elution profiles from the CMSephadex C-25 column. The fractions shadowed were collected and used as the purified venom hyaluronidase preparation.

Hyaluronidase hydrolyzes glycosidic linkages of hyaluronan and eventually produces oligosaccharides. Bovine testes hyaluronidase is the most extensively studied of this class of enzymes and is known to produce primarily tetrasaccharides (31). The bovine enzyme was used in this study and was compared to snake venom hyaluronidase. Hyaluronan was incubated with purified snake venom hyaluronidase at different incubation times of 8, 24, and 48 h. When hyaluronan was incubated for 8 h with purified snake venom hyaluronidase, two major slow moving bands were observed (X and Y of Fig. 4, lane 3). At 48 h digestion, the major fast moving band (X in Fig. 4, lane 5) became more prominent, while the intensities of the other slow moving bands decreased. Slower moving bands are a different degree of oligosaccharides larger than tetrasaccharides. Bovine enzyme digested hyaluronan to give only

trix contortrix is a nonaggregated protein with a minimum apparent molecular weight of 61,000 Da. The molecular weight of purified hyaluronidase from A. contortrix contortrix was determined by two indeTABLE II

Purification of Agkistrodon contortrix contortrix Hyaluronidase

Purification stage

Protein (mg)

Specific activity (unit/mg)

Total activity (unit)

Activity yield (%)

Crude venom Sephacryl S-200 HR CM-Sephadex C-25

500 33.4 0.3

243 (1) 1,316 (5) 67,200 (277)

121,500 43,954 20,160

100.0 36.2 16.6

FIG. 2. Purification of hyaluronidase as shown in an SDS-PAGE profile. The electrophoresis was carried out on PhastSystem with a 10 –15% gradient gel. Lanes 1 and 5, molecular weight markers (size on the left); Lane 2, crude venom; Lane 3, protein components from partially purified snake venom from the first column (shaded area of Fig. 1A); Lane 4, purified hyaluronidase after the second column (from Fig. 1B shaded area).

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FIG. 3. MALDI-TOF mass spectrum of the purified hyaluronidase from A. contortrix contortrix venom.

one major spot following a 24-h incubation (Fig. 4, lane 7). According to the study of Shimada and Matsumura (31), the spot from bovine enzyme action was tetrasac-

FIG. 4. Thin-layer chromatograms of the digestion products of hyaluronan produced by purified snake venom hyaluronidase. Digestion products were developed on the thin-layer plate and oligosaccharides were visualized by orcinol-H 2SO 4 staining. Lane 1, glucuronic acid; Lane 2, hyaluronan; Lane 3, hyaluronan incubated with purified snake hyaluronidase for 8 h; Lane 4, hyaluronan incubated with snake hyaluronidase for 24 h; Lane 5, hyaluronan incubated with snake hyaluronidase for 48 h; Lane 6, hyaluronan incubated with bovine testes hyaluronidase for 8 h; Lane 7, hyaluronan incubated with bovine testes hyaluronidase for 24 h.

charide. The spots produced by the purified snake venom enzyme correspond to the bovine enzyme digestion products. This suggests that snake venom hyaluronidase also produces tetrasaccharides similar to the bovine enzyme. It seemed that the end product of venom hyaluronidase action was tetrasaccharide. To the reaction mixture of hyaluronan and venom enzyme at 48 h, more purified venom enzyme was added. The electrophoretic result indicated that the pattern was similar to X and Y spots shown in Fig. 4, line 5. This indicated that tetrasaccharide did not further degrade to produce disaccharides. In order to confirm that the final product from hyaluronan is tetrasaccharide, the molar ratio of glucuronic acid to reducing terminal units was determined. The amount of reducing units was measured using the Morgan-Elson reaction as modified by Reissig et al. (27). Glucuronic acid residues were measured by the carbazole method of Bitter and Muir (30). A molar ratio of 2 to 1 (glucuronic acid to reducing terminal units) indicates tetrasaccharide products (Fig. 5A). However, if the product is hexasaccharide, then there should be a 3 to 1 ratio (Fig. 5B). Both spots in the thin-layer chromatography (X and Y spots in Fig. 4, lane 5) were analyzed for molar ratio. The spot at X (Fig. 4, spot X) had a ratio of 1.92, indicating it is tetrasaccharide. The spot at Y, however, gave the ratio of 2.98; therefore, we conclude that this spot is hexasaccharide. In order to investigate the substrate specificity of bovine and snake venom hyaluronidase, a number of

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FIG. 5. Chemical structure of tetrasaccharide (A) and hexasaccharide (B). By determining the molar ratio of glucuronic acid to reducing units, one can elucidate the size of the oligosaccharide.

polysaccharides were digested with the enzymes. The experimental results are summarized in Table III. Snake venom hyaluronidase has a narrower substrate specificity than that of bovine enzyme, as the snake venom enzyme hydrolyzed hyaluronan exclusively. In contrast to the snake venom enzyme, bovine hyaluronidase hydrolyzed hyaluronan exclusively. In contrast to the snake venom enzyme, bovine hyaluronidase hydrolyzed hyaluronan, chondroitin, and chondroitin sulfates, A, C, D, and E, without any effect on chondroitin

TABLE III

Substrate Specificity of Snake Venom Hyaluronidase

Name Hyaluronan Chondroitin Chondroitin sulfate A Chondroitin sulfate B Chondroitin sulfate C Chondroitin sulfate D Chondroitin sulfate E Haparin p-Nitrophenyl-Dglucuronide p-Nitrophenyl-Nacetyl-D-glucosaminide a b

⫹, Hydrolyzed. ⫺, No hydrolysis.

Hydrolysis by bovine testes hyaluronidase

Hydrolysis by snake venom hyaluronidase

⫹a ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫺

⫹ ⫺b ⫺ ⫺ ⫺ ⫺ ⫺ ⫺

⫺

⫺

⫺

⫺

sulfate B or heparin. Chromogenic substrates can also be useful for investigating substrate specificity of glycosidases. They are especially useful for characterizing exo-type glycosidases. Hyaluronidase has endo-type glycosidase activity; however, it may also have exo-type activity as well. Therefore, two chromogenic substrates, p-nitrophenyl-␤-D-glucuronide and p-nitrophenyl-N-acetyl-␤-D-glucosaminide were incubated with snake venom hyaluronidase for 24 h. Neither of these substrates was hydrolyzed by snake venom or bovine hyaluronidase. Bacterial hyaluronidase can cleave ␤1,4-glycosidic linkages to produce ⌬4,5 unsaturated glucuronic acid instead of glucuronic acid. The unsaturated glucuronic acid displays an intense absorption at 232 nm. However, no such absorption was observed for snake venom enzyme cleavage products. Therefore, it was concluded that the hydrolysis product of snake venom hyaluronidase was glucuronic acid instead of unsaturated glucuronic acid. Mode of Glycosidic Linkage Cleavage There are two possible tetrasaccharides that can be produced after snake venom hyaluronidase digestion of hyaluronan. One has a glucuronic acid unit at the nonreducing end (Fig. 6A), and the other has an Nacetylglucosamine unit at the nonreducing end (Fig. 5B). The two possible tetrasaccharides can be differentiated by digestion with either ␤-glucuronidase or ␤-Nacetylhexosaminidase.

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by sodium ions, and it reached its maximal activity at 0.2 M sodium ions (Fig. 7C). However, calcium ions did not effect enzyme activity. DISCUSSION

Hyaluronidases are commonly found in nature and some of them have been isolated and characterized. The substrate specificity of hyaluronidase depends on the source. So far, three main types of hyaluronidases have been isolated. The first type produces the main degradation products of ⌬4,5-unsaturated glucuronic acid at the nonreducing end and N-acetylglucosamine at the reducing end. This type of enzyme is found in bacteria (15, 16) and is known as hyaluronan lyase (also eliminase). The second type of hyaluronidase is

FIG. 6. Thin-layer chromatograms of the digestion products of tetrasaccharide with ␤-glucuronidase or ␤-N-acetylhexosaminidase. Tetrasaccharide was incubated with ␤-glucuronidase or ␤-N-acetylhexosaminidase. Lane 1, tetrasaccharide obtained from the digestion of hyaluronan with snake venom enzyme; Lane 2, tetrasaccharide incubated with ␤-glucuronidase; Lane 3, tetrasaccharide incubated with ␤-N-acetylhexosaminidase.

When hyaluronan was digested with the bovine hyaluronidase, the major band in thin-layer chromatography was shown to be tetrasaccharide. The purified snake venom hyaluronidase also produced a major band in thin-layer chromatography that was in the same spot obtained from using bovine enzyme. Thus, the tetrasaccharide obtained from the hydrolysis of hyaluronan from snake venom enzyme digestion was used for linkage cleavage study. When ␤-glucuronidase was used, the tetrasaccharide was hydrolyzed and gave two spots (Fig. 6, lane 2, spots Z and X). No hydrolysis took place when ␤-acetylhexosaminidase was used to digest the tetrasaccharide (Fig. 6, lane 3). This result indicates that the nonreducing terminal of the tetrasaccharide is glucuronic acid (Fig. 5A). This indicates that hydrolysis of the glycosidic linkage in hyaluronan occurs at the ␤1,4- and not at the ␤1,3-bond by snake venom hyaluronidase (Fig. 6). Other Enzymatic Properties The optimum pH for snake venom hyaluronidase was found to be 6.0 and the activity curve is nearly symmetrical (Fig. 7A). At 37°C the enzyme had maximal activity which was lost at 60°C (Fig. 7B). This indicates that at 60°C the enzyme was completely denatured. The enzyme activity was greatly influenced

FIG. 7. The effect of pH (A), temperature (B), and NaCl concentration (C) on hyaluronidase activity.

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an endo-␤-glucuronidase which produces tetrasaccharides with N-acetylglucosamine at the nonreducing end and glucuronic acid at the reducing end. This type of hyaluronidase is found in salivary glands of leeches (14). The third type of enzyme is an endo-␤-N-acetylhexosaminidase and produces glucuronic acid at the nonreducing end and N-acetylglucosamine at the reducing end. This type of enzyme is often found in lysosomes of higher animals and is also present in testicles of higher animals. The hyaluronidase enzyme isolated from A. contortrix contortrix venom belongs to the third type as described above, making it an endo-␤-N-acetylhexosaminidase. The first evidence for this categorization is that the tetrasaccharide product was hydrolyzed by ␤-glucuronidase but not by ␤-N-acetylhexosaminidase. Thus, the cleavage point was at the ␤1,4-glycosidic linkage which produces N-acetylglucosamine at the reducing end. In addition the Morgan-Elson reaction gave a positive result. Only if N-acetylglucosamine is at the reducing terminal will there be a positive reaction using Morgan-Elson’s method. This is additional proof that the reducing end is N-acetylglucosamine. Thus, the snake venom hyaluronidase is more closely related to the bovine enzyme than to the leech enzyme. The bacterial hyaluronidase produces oligosaccharides with ⌬4,5-unsaturated glucuronic acid at the nonreducing end. This compound can be detected spectrophotometrically at 232 nm. This absorption was not observed for the snake venom enzyme. Moreover, ultraviolet absorption spots were not observed on the thin-layer chromatogram. Most bacterial enzymes, with the exception of streptomyces, produce disaccharides as the end product of hyaluronan hydrolysis. However, the snake venom enzyme’s main products were a mixture of hexa- and tetrasaccharides. The bovine enzyme also produces hexa- and tetrasaccharides as the main hydrolysis products. These results indicate that the snake venom hyaluronidase is quite different from the bacterial enzymes. The snake venom enzyme lacks exo-glycosidase activity because it failed to hydrolyze p-nitrophenyl-␤D-glucuronide and p-nitrophenyl-N-acetyl-␤-D-glucosaminide. There is some similarity for bovine and snake venom enzymes but there is also a prominent difference in the two enzymes. The difference can be seen on substrate specificity. Bovine hyaluronidase hydrolyzes chondroitin, chondroitin sulfates A, C, D, and E, besides hyaluronan, the snake venom hyaluronidase is only specific to hyaluronan. The optimum pH for snake venom hyaluronidase was 6.0, while that of bovine testicular enzyme was 5.0 (34). The bovine enzyme also showed a transglycosylation reaction with the optimum pH of 7.0 (34, 35). It is of much interest that some hyaluronidase also possesses transglycosylation (36).

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Although we noted that snake venom hyaluronidase is somewhat similar to bovine enzyme and less similar to the enzyme produced from bacteria that produce disaccharide, there is also some difference from bovine enzyme. Takagi et al. (37) showed that bovine hyaluronidase produced tetrasaccharide because the hexaand disaccharide produced transformed to octamer due to transglycosylation. The octamer was split into half by the action of bovine hyaluronidase. Our result in Fig. 4, lanes 5, 6, and 7, showed that bovine enzyme gave only a tetrasaccharide spot, confirming the result of Takagi et al. (37). Given its selective specificity, snake venom hyaluronidase may become a highly useful tool in cell and tissue biology. ACKNOWLEDGMENT We thank Dr. Y.-T. Li of Tulane University for the valuable advice we received.

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