Hyaluronidase assay using fluorogenic hyaluronate as a substrate

ANALYTICAL

BIOCHEMISTRY

191, 21-24 (1990)

Hyaluronidase Assay Using Fluorogenic Hyaluronate as a Substrate Toshiya Nakamura,* Mitsuo Majima,**t Shinri Tamura,* and Masahiko Endo**l *Department of Biochemistry, Japan, and TKushiro Junior

Received

May

Kohmei

Kubo,*

Hirosaki

036,

2, 1990

Hyaluronidase in mammalian cells is an important enzyme involved in the catabolism of glycosaminoglycans such as hyaluronate and chondroitin sulfates (1). The enzyme activity has generally been measured by determination of N-acetylhexosamine at the reducing terminals of oligosaccharides (2-4), which are the degradation products of the enzyme reaction, using the method of Reissig et al. (5). The above assay method, however, is complicated in comparison with that for bacterial hyaluronidases, such as Streptomyces hyaluronidase, the activities of which are easily detectable by determination of the specific absorption of unsaturated uranic acid at 232 nm (6). Recently, Hase et al. (7) reported a method for detection of sugar chains by pyridylamination of the reducing

0003.2697/90 Copyright All rights

Takagaki,*

Hirosaki University School of Medicine, 5 Zaifu-cho, College, l-IO-42 Midorigaoka, Kushiro 085, Japan

The reducing terminal of hyaluronate was labeled with a fluorogenic reagent, 2-aminopyridine. The pyridylaminohyaluronate was incubated with testicular hyaluronidase for 1 h. After incubation, 4 vol of ethanol was added to the incubation mixture, followed by centrifugation. The fluorescence of the supernatant containing the degradation products of hyaluronidase digestion was then determined by fluorospectrophotometry (excitation wavelength, 320 nm; emission wavelength, 400 nm). It was found that the increase of the pyridylamino products was linearly correlated with enzyme concentration (up to 0.1 national formulary unit), incubation time (up to 60 min), and substrate concentration (up to 2.6 PM). The fluorogenic substrate was also applicable for the determination of crude hyaluronidase. This simple, rapid, and sensitive hyaluronidase assay was made possible by the use of pyridylaminohyaluronate as a substrate. 0 1990 Academic PWS, Inc.

’ To whom

Keiichi

correspondence

should

$3.00 0 1990 by Academic Press, of reproduction in any form

be addressed.

terminals of N-glycosidically linked oligosaccharides, and this method has been useful for analysis of the structure of sugar chains of glycoproteins (8-11). This method is also applicable for the structural analysis of glycosaminoglycans (12). This report describes a simple and sensitive hyaluronidase assay using pyridylaminohyaluronate (PA-hyaluronate)’ as a substrate. MATERIALS

AND

METHODS

Materials. 2-Aminopyridine and sodium cyanoborohydride were purchased from Wako Chemical Co. (Osaka, Japan) and Aldrich Chemical Co. (Milwaukee, WI), respectively. Standards of PA-monosaccharides were the same as described previously (12). Hyaluronate was prepared from human umbilical cord by the method of Danishefsky and Bella (13), followed by further purification by Dowex l-X2 chromatography. Hyaluronate tetra- and hexasaccharides obtained by testicular hyaluronidase digestion were the same as described previously (14), and hyaluronate octa-, deca-, and dodecasaccharides were prepared by gel filtration of testicular hyaluronidase digests on a Bio-Gel P-4 column according to the method of Cowman et al. (15). Bovine testicular hyaluronidase Type I-S (EC 3.2.1.35) and bovine epididymis /3-N-acetylglucosaminidase A (EC 3.2.1.30) were purchased from Sigma Chemical Co. (St. Louis, MO). Exo-fi-glucuronidase (no activity of exo-pN-acetylglucosaminidase) was purified from rabbit liver by using p-nitrophenyl-B-D-glucuronide as a substrate according to the method described previously (16). Sephadex G-15 was purchased from Pharmacia LKB Biotechnology Inc. (Uppsala, Sweden). Other reagents and chemicals were obtained from commercial sources.

* Abbreviations unit.

used:

PA, pyridylamino;

NFU,

national

formulary

21 Inc. reserved.

22

NAKAMURA

Preparation of PA-hyaluronate as substrate. Pyridylamination of hyaluronate was performed by the method of Hase et al. (7). Briefly, to the hyaluronate (5 mg) was added 2-aminopyridine in 1.5 ml of hydrochloric acid. The pH of the solution had been adjusted to 6.2 after dilution with 2 vol of water. The mixture was heated at 100°C for 13 min in a sealed tube. The tube was then opened and 90 ~1 of the reducing reagent (prepared by mixing 29 mg of sodium cyanoborohydride, 40 ~1 of 2-aminopyridine solution, and 50 ~1 of water) was added. The tube was resealed and heated at 90°C for 15 h. The tube was reopened, and 4 vol of ethanol was added to the reaction mixture, which was then left to stand for 30 min at O”C, followed by centrifugation. The precipitate was redissolved in 1 ml of water, and the above procedure (ethanol precipitation) was repeated five times. Most of the free 2-aminopyridine was removed by repeated ethanol precipitation. Then, the reaction product was applied to a column (1.0 X 60 cm) of Sephadex G-15, which was equilibrated and eluted with 0.1 M acetic acid, and 1.8-ml fractions were collected. Fluorescence of each fraction was measured by a fluorescence spectrophotometer with an excitation wavelength of 320 nm and an emission wavelength of 400 nm. Uranic acid was determined by the method of Bitter and Muir (17). The fractions showing fluorescence and containing uranic acid were collected, evaporated to dryness, and used as PA-hyaluronate. The total recovery of PA-hyaluronate was 7981%. Because hyaluronate is difficult to dissolve, the hyaluronate solution must be stirred carefully to ensure complete dissolution when making up a stock solution. PA-hyaluronate in stock solution is stable for at least 2 months when kept in the dark at -20°C. The PA-hyaluronate was subjected to gel filtration on a column (0.8 X 30 cm) of Shodex OHpak KB-803 (Showa Denko Co. Ltd., Tokyo, Japan), which was prepacked with poly(hydroxymethacrylate)-type resin for high-performance size-fractionation chromatography. HPLC was carried out at a flow rate of 0.5 ml/min at 30°C using 0.2 M NaCl as an eluent. The elution profiles, which were monitored by both a uv detector (Hitachi L-4200) and a fluorescence detector (Hitachi F-1050), overlapped, indicating that hyaluronate was pyridylaminated (Fig. 1B). However, hyaluronate was degraded slightly after pyridylamination (Figs. 1A and 1B). The reducing terminal sugar residue of the PAhyaluronate was identified as N-acetylglucosamine by the method described previously (12). Hyaluronate oligosaccharides (tetra-, hexa-, octa-, deca-, and dodecasaccharides) were also pyridylaminated as above, and then the reaction mixtures were applied individually to a column of Sepbadex G-15. Both fluorescenceand uranic acid-positive fractions were collected and used as PA-oligosaccharides. Quantification of PA derivatives was performed by comparison of the intensity of fluorescence with that of a pyridylamino-N-acetylglucosamine standard (500 pmol/ml).

ET

AL.

0

5 10 15 20 Elution time (mid

25

FIG. 1.

HPLC of hyaluronate before and after pyridylamination. Hyaluronates before (A) and after (B) pyridylamination were chromatographed on a column (0.8 X 30 cm) of Shodex OHpak KB-803. The chromatographic conditions are described in the text. (-) Fluorescence with an excitation wavelength of 320 nm and emission wavelength of 400 nm; (- - -) absorbance at 210 nm.

Enzyme assay. Hyaluronidase assay using PA-hyaluronate was performed as follows. A reaction mixture (50 ~1) containing 50 pmol (1.5 pg) of PA-hyaluronate and enzyme protein (0.1 NFU) in 50 mM sodium acetate buffer, pH 5.0, containing 0.15 M NaCl was incubated at 37°C for 1 h. To the reaction mixture was added exactly 200 ~1 of ethanol saturated with NaCl. The mixture was left to stand for 30 min at 0°C followed by centrifugation. To 100 ~1 of the supernatant was added 300 ~1 of 0.5 M sodium acetate buffer, pH 4.0. The fluorescence of the solution was measured with a fluorescence spectrophotometer (Hitachi 204-R) with an excitation wavelength of 320 nm and an emission wavelength of 400 nm. /3-Glucuronidase digestion of hyaluronidase digests was performed as described previously (16). P-N-Acetylglucosaminidase digestion was performed by the method of Tarentino and Maley (18). Identification of ethanol-soluble PA-oligosaccharides in hyaluronidase digests. Chain lengths and nonreducing terminal sugars of the PA-oligosaccharides in hyaluronidase digests, which were recovered in the soluble fraction of 80% ethanol, were determined by HPLC using a column of Shodex OHpak KB-803 under the same conditions as described above. Nonreducing terminal sugars of PA-oligosaccharides were also identified by using the same HPLC conditions as those described above after exoglycosidase digestion. RESULTS

Digestion

of PA-hyaluronute with Hyaluronidase PA-hyaluronate was incubated at 37°C with testicular hyaluronidase at pH 5.0. To the reaction mixture was added 4 vol of ethanol saturated with NaCl, and the fluorescence of the supernatant was measured. It was found that the increase of PA products was linearly correlated with the amount of enzyme (up to 0.1 NFU,

HYALURONIDASE

6

ASSAY

USING

FLUOROGENIC

23

HYALURONATE

Oh-sup

Oh-ppt

A

2 I

40 Lzl 0

26 ,P

0.03 0.06 Enzyme (N.F.U.)

0.09

lh-sup

1h-ppt

B

9;4 s pz 2 .” B B so 0

16

Ph-sup

2h-ppt 2 15

30 45 Time (mid

60

Jk Jh-sup

Jh-ppt

.

c

12 8 4 u OO

1

0

2

10 20 0 Elution time (mm)

10

Substrate@Ml FIG. 2.

Determination of hyaluronidase activity. The conditions of the enzyme reaction are described in the text with the following modifications: (A) effects of enzyme concentration; (B) effects of incubation time; (C) effects of substrate concentration.

20

FIG. 3.

HPLC of hyaluronidase digests. PA-hyaluronate was incubated for 1, 2, or 3 h under standard conditions. The digests were fractionated with ethanol, and the soluble and insoluble fractions were subjected to HPLC. The chromatographic conditions are described in the text: ppt, insoluble fraction; sup, soluble fraction; 0 h, 1 h, 2 h, and 3 h indicate incubation time.

Fig. 2A), incubation time (up to 60 min, Fig. 2B), and substrate concentration (up to 2.5 PM, Fig. 2C). Chain Length of PA Products by Hyaluronidase Digests PA-hyaluronate was incubated with testicular hyaluronidase for 1,2, or 3 h. Ethanol-soluble and -insoluble fractions of the reaction products were subjected to HPLC by gel filtration. The amounts of ethanol-insoluble products decreased gradually and a shift of the peak was shown toward a larger elution volume as the period of incubation increased (Fig. 3). On the other hand, ethanol-soluble products increased, being represented as two peaks with retention times of 16.6 and 17.0 min (Fig. 3). The chain lengths of these oligosaccharides were estimated to be hexa- and octasaccharides, respectively, in comparison with the retention times of PA-oligosaccharide standards (Fig. 4). Identification of Nonreducing Terminal Sugars of Ethanol-Soluble PA-oligosaccharides The ethanol-soluble PA-oligosaccharides were subjected to HPLC after incubation with P-glucuronidase or @-N-acetylglucosaminidase A. Only @-glucuronidase digestion caused shifts of the two peaks (Fig. 4), suggesting that the nonreducing terminal sugars were glucuranic acid. Incubation of PA-hyaluronate with an Enzyme Fraction from Rabbit Liver The applicability of the new method to a crude hyaluronidase preparation was investigated. The crude en-

zyme was obtained from a rabbit liver extract by ammonium sulfate fractionation. The protein emerging within the range of 30-50% saturation was used as the crude enzyme fraction. PA-hyaluronate was incubated with the enzyme fraction, and the enzyme reaction was

0

10

15 Elution time (mid

20

FIG. 4. HPLC of ethanol-soluble PA-oligosaccharides in hyaluronidase digests. (A) Control; (B) after incubation with P-glucuronidase; (C) after incubation with /3-N-acetylglucosaminidase A. Arrows indicate the elution positions of the standards of PA-hyaluronate oligosaccharides: 12, dodecasaccharide; 10, decasaccharide; 8, octasaccharide; 6, hexasaccharide; 4, tetrasaccharide. The chromatographic conditions were the same as in the legend of Fig. 1.

24

NAKAMURA

Time (min)

FIG. 5.

Time course of crude hyaluronidase activity using PA-hyaluronate as a substrate. The crude enzyme fraction was obtained from a rabbit liver extract by ammonium sulfate fractionation. PAhyaluronate was incubated with the enzyme fraction in 50 mM sodium acetate buffer, pH 5.0, containing 0.15 M NaCl at 37°C for 1 h. After incubation, hyaluronidase activity was assayed as described in the text.

found to be linear with time for at least 60 min under standard conditions (Fig. 5), indicating that the new assay method was applicable to crude enzyme. DISCUSSION Recently, Takagaki et al. (19) purified endo-@xylosidase from the mid-gut gland of Patnopecten using a fluorogenic substrate, a chondroitin/dermatan sulfate chain bearing 4-methylumbelliferyl xyloside at the reducing terminal (20). In addition, pyridylamination of the sugar chain of N-glycosidically linked oligosaccharides (7-11) was applied to preparations of fluorogenic glycosaminoglycans (12), and the activity of endo-&galactosidase (21) was detectable by using PA-chondroitin sulfate as a substrate (22). The use of a fluorogenic substrate made determination of the activities of the endoglycosidases rapid and sensitive. Determination of the activity of hyaluronidase has previously been complicated and troublesome, but, as reported here, application of the fluorogenic hyaluronate made determination of the enzyme activity simple and sensitive. The main products of hyaluronidase digestion are known to be tetra- and hexasaccharides (2), whereas the major PA products of hyaluronidase digestion in this study were shown to be hexa- and octasaccharides (Fig. 4). This difference may have been due to the presence of 2-aminopyridine at the reducing terminal. Although the reaction products appeared to contain tetra- and hexasaccharides without fluorescence, as well as PA-hexa- and PA-octasaccharides derived from the reducing terminal site of PA-hyaluronate, these nonfluorescent oligosaccharides are not. detectable by this method. The fluorometric assay reported here has the following characteristics. (i) In a calorimetric assay, the sensitivity of the method is better than 15 NFU (23). In contrast, the fluorogenic method allows assay with less than 0.1 NFU (Fig. 2A), suggesting that the method is at least 150 times more sensitive than the calorimetric as-

ET

AL.

say. (ii) The assay procedures are very simple, requiring only the addition of ethanol. (iii) Calorimetric assays involving chemical reactions are sometimes affected by compounds in the crude enzyme preparation. In contrast, the fluorogenic method is also applicable for determination of the activity of a crude enzyme without such interference. The fluorogenic hyaluronate was considered to be useful for the determination of hyaluronidase activity in various tissues, the purification of the enzyme, and also for the study of hyaluronate catabolism. REFERENCES 1. Rod&n, L. (1980) in The Biochemistry of Glycoproteins and Proteoglycans (Lennarz, W. J., Ed), pp. 267-371, Plenum, New York. 2. Aronson, N. N., and Davidson, E. A. (1967) J. Biol. Chem. 242, 441-444. 3. Cashman, D. C., Laryea, J. U., and Weissmann, B. (1969) Arch. B&hem. Biophys. 135,387-395. 4. Orkin, 1042.

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18. Tarentino, A. L., and Maley, F. (1972) in Methods in Enzymology (Ginsburg, V., Ed.), Vol. 28, pp. 772-776, Academic Press, San Diego, CA. 19. Takagaki, K., Kon, A., Kawasaki, H., Nakamura, T., Tamura, S., and Endo, M. (1990) J. Biol. Chem. 265,854~860. K., Kon, A., Kawasaki, H., Nakamura, T., and Endo, 20. Takagaki, M. (1990) J. Biochem. Biophys. Methods 19,207-214. K., Nakamura, T., and Endo, M. (1988) B&him. 21. Takagaki, Biophys. Acta 966,94-98. 22. Takagaki, K., Kon, A., Kawasaki, H., Nakamura, T., Tamura, S., and Endo, M. (1990) Biochem. Biophys. Res. Commun. 169,1521. 23. Linker, A. (1974) in Methods of Enzymatic H. U., Ed), Vol. 2, pp. 944-948, Academic

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