277
Clinica Chimica Acta, 95 (1979) 277-284 @ Elsevier/North-Holland Biomedical Press
CCA 1051
A NEW MICRO-ASSAY
FOR HYALURONIDASE
S.S. CHEN a**, D.S. HSU a and P. HOFFMAN
ACTIVITY
b
a Department of Obstetrics and Gynecology and b Department of Orthopedic Surgery, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794 (U.S.A.) (Received
November
2&h,
1978)
Summary A new micro-method for the assay of hyaluronidase activity is described. The method utilizes chondroitin sulfate as a substrate. The degraded products of chondroitin sulfate by hyaluronidase are determined by modified disc gel electrophoresis. The products are first concentrated into a single band in acrylamide gel, and then the band is stained with cetylpyridinium chloride. The absorbance of the band is proportional to the amount of the degraded products. The hyaluronidase activity is therefore linearly related to the absorbance. This procedure represents a sensitive method for the assay of hyaluronidase activity in the serum and urine.
Introduction Hyaluronidase which reduces the viscosity of hyaluronic acid has been demonstrated in skin [ 11, spleen [ 21, urine [3,4] and plasma [ 5,6]. It has been found ubiquitously to be present in mammalian tissues, synovial fluid and serum. The assay method used by others for hyaluronidase [7] measures the amount of terminal N-acetylglucosamine liberated from hyaluronic acid. Hyaluronidase activity has been found to be elevated in sera of patients with atherosclerosis associated with hypertension [ 81 and rheumatoid arthritis with depolymerization of hyaluronic acid and increased permeability of connective tissues [g-11]. By contrast, hyaluronidase activities in the sera of cancer patients are found to be lower than normal [ 121. The physiological significance of hyaluronidase in sera is not clearly defined at present. This paper described a method which will be useful in these studies.
* Correspondence 11040.
U.S.A.
should
be
addressed
to:
Dr.
S.S.
Chen.
270-05,
76th
Avenue,
New
Hyde
Park,
NY
278
We have devised a method using disc gel electrophoresis to concentrate the degraded products of chondroitin sulfate by hyaluronidase to a discrete band. The band is stained with cetylpyridinium chloride and the amount is quantitated by scanning the band with a spectrophotometer. Using this method, the levels of hyaluronidase activities in sera could be easily determined. Materials and methods Alkali-treated bovine nasal chondroitin 4-sulfate used as a substrate for hyaluronidase determination was prepared as previously described [ 131. Hyaluronidase (Lot No. 60572, bovine testis), which is the National Formularly Reference Standard, was obtained from the American Pharmaceutical Association. It was compressed with lactose to a tablet form. The tablet contained 8.8 National Formularly Standard (NF) units/mg. Hyaluronidase from human umbilical cord was obtained from Mann Research Laboratories, Inc. Comparative assays for the standard hyaluronidase were performed with turbidity reducing and colorimetric methods. The turbidity reducing method was carried out as described by Tolksdorf et al. [14]. The formation of N-acetylglucosamine was monitored according to the procedure of Bollet et al. [ 71. Glucuronic acid was determined by carbazole method [ 151. Procedure for hyaluronidase assay For the assay of the activity of hyaluronidase obtained from the American Pharmaceutical Association, the reaction was carried out in 0.1 mol/l phosphate buffer, pH 5.3, containing 0.15 mol/l NaCl and 10 mg chondroitin sulfate per ml. The reaction was terminated by heating for 5 min in boiling water. After cooling, a 20 ~1 aliquot was mixed with 5 ~1 of 40% sucrose in order to increase the density and applied on the gel for electrophoresis. For the determination of serum hyaluronidase activity, 50 ~1 of serum was mixed with 0.2 ml of acetate buffer, pH 3.36, containing NaCl and chondroitin sulfate. The final reaction mixture contained 0.1 mol/l acetate, 0.15 mol/l NaCl, and 4 mg/ml chondroitin sulfate. The mixture was incubated at 37°C for 18 h. The reaction was terminated by heating the mixture for 5 min in boiling water. After cooling and centrifugation, 50 ~1 of the supernatant solution was mixed with 5 ~1 of 40% sucrose and applied on the gel for electrophoresis. A unit of the enzyme activity was expressed as 1 pg of degraded chondroitin sulfate produced in 18 h at 37°C. Standard of degraded chondroitin sulfate The standard of degraded chondroitin sulfate was prepared by incubating chondroitin sulfate with excess amount of hyaluronidase for 18 h at 37°C. The hyaluronidase was then inactivated by heating for 5 min in boiling water. This degraded product gave a single band running with the buffer front in the disc gel electrophoresis. The amount was determined by quantitation of glucuronic acid by the carbazole method and converted to the amount of chondroitin sulfate. For each set of runs 50 ~1 containing 20 pg of the degraded chondroitin sulfate was applied on a gel for electrophoresis to serve as a standard.
219
Disc gel electrophoresis for the determination of the degraded chondroitin sulfate The disc gel electrophoresis was done using ethanolamine/HCl/borate system as previously described [ 131. The gel was prepared containing 7.5% acrylamide in ethanolamine/HCl/ borate buffer. This buffer was prepared as four times the required final concentration by mixing 3.8 ml ethanolamine and 2 ml concentrated HCl to a total volume of 100 ml. The reservoir buffers contained 4.7 g boric acid and 2.5 ml ethanolamine in 2 1. The gel was left at room temperature overnight to decompose the excess amount of ammonium persulfate which otherwise would also form a precipitate with cetylpyridinium chloride, interfering with the test result. After the gels were set up in the electrophoresis apparatus and tubes and upper reservoirs were filled with buffer, the samples were applied on the top of the gels, and the electrophoresis was run at 200 V with 2.5 mA/tube for 5 min and then at 5 mA/tube for 20 min. After electrophoresis, the gels were stained in 1% cetylpyridinium chloride containing 0.025 mol/l ethylenediamine tetraacetic acid for 90 min. They were scanned on a Gilford gel scanner at 360 nm. Results Degradation of chondroitin sulfate by hyaluronidase The alkali-treated chondroitin sulfate used as the substrate for hyaluronidase assay contains large molecular of varying chain size. As shown in Fig. 1, they migrated in the gel as a broad band. By incubating with hyaluronidase the chondroitin sulfate was hydrolyzed to smaller molecules with diminished chain size. They were separated into 2 parts by the modified disc gel electrophoresis. One part which consisted of the smaller chains were concentrated into a thin band and migrated with the buffer front, giving a sharp peak after staining the gel with cetylpyridinium chloride and scanning the gel by the spectrophotometer. The other part was the mixture of the undegraded and the partially
o&---2
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4
5
LENGTH OF GEL (cm) Fig. 1. Disc gel electrophoresis of chondroitin sulfate. A mixture of 200 fig of chondroitin sulfate and 0.25 NF units of heat inactivated hyaluronidase was run in a gel. After the gel was stained in cetylpyridinium chloride, the whole gel was scanned at 360 nm.
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LENGTH OF GEL b-n) Figs. 2 and 3. Disc gel eleetroghoresis of chondroitin sulfate after partially degradation by hyaluronidase. Chondroitin sulfate (4 me/ml) was incubated with commercial hyaluronidase, 5 NF units/ml. Fifty 1.11of the incubating mixture were drawn at various times, run in a gel and scanned at 360 nm after staining with cetylpyridinium chloride. Fig. 2 shows the result of 20 min incubation, and Fig. 3, SO min. The sharp peaks are the chondroitin sulfate oligosaccharides with low molecular weights concentrated to a single discrete band, and run with the buffer front in the gel electrophoresis.
degraded chondroitin sulfates which migrated as a broad peak behind and separated from the sharp peak. The results of the scanning profiles of the degraded products after incubation for 20 min and 90 min are shown in Fig. 2 and Fig. 3, respectively. The absorbance of the sharp band increases as the time of incubation increases. Fig. 4 shows that the absorbance is linearly proportions to the incubation time; thus the degraded products increase with time of hydrolysis.
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MINUTES INCUBATION Fig. 4. Degradation of chondroitin sulfate by hyaluronidase and the duration of incubation. One hundred ~1 containing 600 ~g of chondroitin sulfate in 0.1 mol/l phosphate buffer, pH 5.3, and 0.16 mol/l NaCl was incubated at 37’C with 0.06 NF units of hyaluronidase for different time intervals. The reaction was stopped by heating in a water bath for 5 min. Twenty ~1 of each solution was used for electxophoresis.
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20
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HYALURONIDASE DEGRADED C~NDROITIN SUt_FATE (pg)
35 NF UNITS OF HYA~URONIDASE (U/ml)
Fig. 5. Linear relationship of the hyaluronidase degraded chondroitin sulfate and absorbance. The hyaluronidase degraded chondroitin sulfate used as a standard for enzyme assay gave a single concentrated band running with the buffer front in the gel electrophoresis. The quantities are linearly related to the absorbance of the peaks. The four peaks shown in the figure represent 13.6 ng, 20.4 tig. 27.2 A% and 34 Pg of the standard chondroitin sulfate, respectively. Fig. 6. Degraded product of chondroitin sulfate versus hyaluronidase concentration. The reaction mixture in a volume of 0.2 ml containing 2 mg ehondroitin suffate, 0.1 moi/i phosphate buffer. pH 5.3, 0.13 moifl NaCI. and different concentration of hyaluronidase was incubated at 37’C for 1 h. The reaction was terminated by heating the reaction mixture in a water bath for 5 min. Twenty pl of each reaction mixture was then assayed for the degraded chondroitin sulfate by gel electrophoresis.
Relationship between the absorbance and the degraded ~h~ndr~~tin sulfate The degraded products obtained by treating the chondroitin sulfate with excess amount of hyaluronidase gave a single sharp band on disc gel electrophoresis. The amount of the degraded chondroitin sulfate is linearly related to the optical density of the peak. This is illustrated in Fig. 5, the degraded chondroitin sulfate between 10 pug and 35 t_lg gave a linear relationship with absorbance. Relationship between hyaluronidase activity and hyaluronidase concentration When the concentration of hyaluronidase was varied, the amount of the degraded chondroitin sulfate produced as determined by the disc gel electrophoresis was linearly proportional to the activity of hyaluronidase. The results are shown in Fig. 6. The detail of the experimental procedure is described in the legend of the figure. From the data of this figure and using the curve in Fig. 5 as a standard, it is calculated that 1 NF unit of hyaluronidase is equivalent to 1216 units of hyaluronidase activity defined in this paper. Comparatiue assays for standard hyaluronidase (NF) by turbidity reducing method, ~o~orimetr~e technique and proposed mi~r~~ssay In the same experimental condition, one NF unit of hyaluronidase from the American Pharmaceutical Association represented 0.35 turbidity reducing unit, 12.2 pg of N-acetylglucosamine demasked by calorimetric technique and 38 pg of chondroitin sulfate degraded by our micro-assay. The experimental condition was at pH 5.3 and 37”C, and in 30 min of reaction time.
282
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3
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5
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8
PH Fig. 7. pH optimum of hyaluronidase from different sources. The solid line shows the pH optimum curve for a crude extract of rat testes, the dotted line for the commercial hualuronidase. The buffers used are phosphate buffer (closed circle) and acetate buffer (open circle).
pH optimum
of hyaluronidase
from different
sources
As shown in Fig. 7, the commercially available testicular hyaluronidase showed an optimal reaction at pH 6. A crude extract from rat tested showed an optimal reaction at pH 4.8. While the activities in human serum and urine tested at pH 6 using phosphate buffer or at pH 4.8 using acetate buffer showed a little activity, maximal activity was obtained when the pH of the acetate buffer was adjusted to pH 3.4. The pH at the end of incubation was about pH 4. Similar result was obtained when lactic acid instead of acetic acid was used as a buffer. Preliminary
observation
of hyaluronidase
activity in human serum
Sera were obtained from patients with different diseases, as well as from normal. The level of hyaluronidase activity tested on 26 normal subjects was fairly constant. The average was 892 + 64 units/ml serum. Control data from the disease states are being collected and the results will be reported in a separate paper. Discussion The crux of this method is based on the observation that the degraded products of chondroitin sulfate also precipitate with cetylpyridinium chloride in the gel at an optimal concentration of cetylpyridinium chloride. Furthermore, the degraded products of chondroitin sulfate can be concentrated to a thin single band by the modified disc gel electrophoresis and separated from the intact or partially degraded chondroitin sulfate. In our buffer system, we used borate as a trailing anion. Borate ion has an electrophoretic mobility lower than that of chondroitin sulfate. Thus, in the gel smaller size chondroitin sulfate will be con-
283
tinuously concentrated into a single band and moves in the gel with the buffer front between the leading anion, Cl-, and the trailing anion, borate. While the larger size chondroitin sulfate will be retarded by the gel leaving behind the buffer front and separated according to their size as a broad band shown in Figs. 2 and 3. The chain size of chondroitin sulfate which will not be subjected to the sieving action of 7.5% acrylamide gel is yet to be determined. However, from our previous experiment [ 131, it is estimated to be less than four tetrasaccharide units. In order to increase the sensitivity of the method, one can use lower concentration of acrylamide gel to increase the pore size. Thus, more’of the degraded products will be concentrated in the sharp band. At present hyaluronidase can be determined by turbidimetric, viscosimetric and calorimetric methods. Turbidimetric and viscosimetric methods are unsatisfactory in clinical use [ 121. Only the calorimetric method is widely used by many investigators [ 7,8,20,21]. Even though the micro-assay proposed is slightly cumbersome compared to calorimetric method, we find that our microassay has potential clinical use in assaying biological fluid and tissue. Since a hyaluronidase degraded chondroitin sulfate system is employed, our method would be less susceptible to any interference. Substances which might interfere with the system are nucleic acid and acid glycosaminoglycans. This interference can be prescreened by gel electrophoresis. Furthermore, any sample containing colored substance can be determined with our method accurately. The procedure described has several distinct advantages. First, chondroitin sulfate is easily obtainable in a pure form and in large quantities. Secondly, hyaluronidase from most bacteria can be eliminated [ 161, and thirdly, our sensitivity can be enhanced greatly by using isotope labelled chondroitin sulfate. Two types of hyaluronidase are known to exist in mammalian tissues. They are lysosomal and testicular types. Each type has its distinct pH optimum. Lysosomal hyaluronidase has pH optimum of 3.5, whereas testicular type, 4.5 to 6.0 [ 171. We note that human hyaluronidase has similar pH optimum as of lysosomal origin, as reported by others [ 7,8,19]. Finally, we believe that the proposed new micro-assay for human hyaluronidase using our chondroitin sulfate-gel electrophoresis system can complement the existing simpler calorimetric methods by overcoming interferences. References 1 Mayer, R.L., Kocholaty. W. and Stanton, D. (1948) Proc. Sot. Exptl. Biol. Med. 67. 529-531 2 Meyer. K. and Rapport, M.M. (1952) in Advances in Enzymology. Vol. 13 (Nerd, F.F., ed.), pp. 199236, Interscience Publishers, New York 3 Ginetzinsky. A.G. (1958) Nature 182.1218-1219 4 Berlyne. G.M. (1960) Nature 185, 389-390 5 Knudsen, P.J. and Koefoed, J. (1961) Nature 191,1306-1307 6 Cobbin. L.B. and Dicker, S.E. (1962) J. Physiol. 163.168-174 7 BoUet, A.J., Banner, W.M., Jr. and Nance, J.L. (1963) J. Biol. Chem. 238. 3522-3527 8 Sempinska, E., Keibasinska. E.. Kostka, B. and Mazurowa, A. (1976) Acta Med. Pal. 17. 79-84 9 Pigman, W., Fabianek. J.. Herp, A., Root, L. and &lick. A. (1963) Arthritis Rheum. 6,788 10 Banner, W.M., Jr. and Cantey, E.Y. (1966) Clin. Chim. Acta 13.746-752 11 Salkii. M.L., Hannah, C.L. and McNeil, E.M. (1976) Clin. Biochem. 9.184-187 12 Nothup, S.N., Stasiw. R.O. and Brown. H.D. (1973) Clin. Biochem. 6,220-228 13 Hsu, D.S., Hoffman, P. and Mashburn. T.A.. Jr. (1973) Anal. Biochem. 52. 382-394 14 Tolksdorf, S., M&ready, M.H.. McCullagh, D.R. and Schwenk, E. (1949) J. Lab. Clin. Med. 34. 7489
284
15 Bitter, T. and Muir. H.M. (1962) Anal. Biochem. 4.330-334 16 Meyer. K. (1971) in The Enzymes. Vol. 5 (Bayer, P.D.. ed.), pp. 307-320, Academic and London 17 Aronson. N.N.. Jr. and Davidson, E.A. (1967) J. Biol. Chem. 242. 437-444 18 DeSalegui, M. and Pigman, W. (1967) Arch. Biochem. Biophys. 120. 60-67 19 Bowness, J.M. and Tan. Y.H. (1968) Biochim. Biophys. Acta 151,288-290
Press. New York
Clinica Chimica Acta, 95 (1979) 277-284 @ Elsevier/North-Holland Biomedical Press
CCA 1051
A NEW MICRO-ASSAY
FOR HYALURONIDASE
S.S. CHEN a**, D.S. HSU a and P. HOFFMAN
ACTIVITY
b
a Department of Obstetrics and Gynecology and b Department of Orthopedic Surgery, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794 (U.S.A.) (Received
November
2&h,
1978)
Summary A new micro-method for the assay of hyaluronidase activity is described. The method utilizes chondroitin sulfate as a substrate. The degraded products of chondroitin sulfate by hyaluronidase are determined by modified disc gel electrophoresis. The products are first concentrated into a single band in acrylamide gel, and then the band is stained with cetylpyridinium chloride. The absorbance of the band is proportional to the amount of the degraded products. The hyaluronidase activity is therefore linearly related to the absorbance. This procedure represents a sensitive method for the assay of hyaluronidase activity in the serum and urine.
Introduction Hyaluronidase which reduces the viscosity of hyaluronic acid has been demonstrated in skin [ 11, spleen [ 21, urine [3,4] and plasma [ 5,6]. It has been found ubiquitously to be present in mammalian tissues, synovial fluid and serum. The assay method used by others for hyaluronidase [7] measures the amount of terminal N-acetylglucosamine liberated from hyaluronic acid. Hyaluronidase activity has been found to be elevated in sera of patients with atherosclerosis associated with hypertension [ 81 and rheumatoid arthritis with depolymerization of hyaluronic acid and increased permeability of connective tissues [g-11]. By contrast, hyaluronidase activities in the sera of cancer patients are found to be lower than normal [ 121. The physiological significance of hyaluronidase in sera is not clearly defined at present. This paper described a method which will be useful in these studies.
* Correspondence 11040.
U.S.A.
should
be
addressed
to:
Dr.
S.S.
Chen.
270-05,
76th
Avenue,
New
Hyde
Park,
NY
278
We have devised a method using disc gel electrophoresis to concentrate the degraded products of chondroitin sulfate by hyaluronidase to a discrete band. The band is stained with cetylpyridinium chloride and the amount is quantitated by scanning the band with a spectrophotometer. Using this method, the levels of hyaluronidase activities in sera could be easily determined. Materials and methods Alkali-treated bovine nasal chondroitin 4-sulfate used as a substrate for hyaluronidase determination was prepared as previously described [ 131. Hyaluronidase (Lot No. 60572, bovine testis), which is the National Formularly Reference Standard, was obtained from the American Pharmaceutical Association. It was compressed with lactose to a tablet form. The tablet contained 8.8 National Formularly Standard (NF) units/mg. Hyaluronidase from human umbilical cord was obtained from Mann Research Laboratories, Inc. Comparative assays for the standard hyaluronidase were performed with turbidity reducing and colorimetric methods. The turbidity reducing method was carried out as described by Tolksdorf et al. [14]. The formation of N-acetylglucosamine was monitored according to the procedure of Bollet et al. [ 71. Glucuronic acid was determined by carbazole method [ 151. Procedure for hyaluronidase assay For the assay of the activity of hyaluronidase obtained from the American Pharmaceutical Association, the reaction was carried out in 0.1 mol/l phosphate buffer, pH 5.3, containing 0.15 mol/l NaCl and 10 mg chondroitin sulfate per ml. The reaction was terminated by heating for 5 min in boiling water. After cooling, a 20 ~1 aliquot was mixed with 5 ~1 of 40% sucrose in order to increase the density and applied on the gel for electrophoresis. For the determination of serum hyaluronidase activity, 50 ~1 of serum was mixed with 0.2 ml of acetate buffer, pH 3.36, containing NaCl and chondroitin sulfate. The final reaction mixture contained 0.1 mol/l acetate, 0.15 mol/l NaCl, and 4 mg/ml chondroitin sulfate. The mixture was incubated at 37°C for 18 h. The reaction was terminated by heating the mixture for 5 min in boiling water. After cooling and centrifugation, 50 ~1 of the supernatant solution was mixed with 5 ~1 of 40% sucrose and applied on the gel for electrophoresis. A unit of the enzyme activity was expressed as 1 pg of degraded chondroitin sulfate produced in 18 h at 37°C. Standard of degraded chondroitin sulfate The standard of degraded chondroitin sulfate was prepared by incubating chondroitin sulfate with excess amount of hyaluronidase for 18 h at 37°C. The hyaluronidase was then inactivated by heating for 5 min in boiling water. This degraded product gave a single band running with the buffer front in the disc gel electrophoresis. The amount was determined by quantitation of glucuronic acid by the carbazole method and converted to the amount of chondroitin sulfate. For each set of runs 50 ~1 containing 20 pg of the degraded chondroitin sulfate was applied on a gel for electrophoresis to serve as a standard.
219
Disc gel electrophoresis for the determination of the degraded chondroitin sulfate The disc gel electrophoresis was done using ethanolamine/HCl/borate system as previously described [ 131. The gel was prepared containing 7.5% acrylamide in ethanolamine/HCl/ borate buffer. This buffer was prepared as four times the required final concentration by mixing 3.8 ml ethanolamine and 2 ml concentrated HCl to a total volume of 100 ml. The reservoir buffers contained 4.7 g boric acid and 2.5 ml ethanolamine in 2 1. The gel was left at room temperature overnight to decompose the excess amount of ammonium persulfate which otherwise would also form a precipitate with cetylpyridinium chloride, interfering with the test result. After the gels were set up in the electrophoresis apparatus and tubes and upper reservoirs were filled with buffer, the samples were applied on the top of the gels, and the electrophoresis was run at 200 V with 2.5 mA/tube for 5 min and then at 5 mA/tube for 20 min. After electrophoresis, the gels were stained in 1% cetylpyridinium chloride containing 0.025 mol/l ethylenediamine tetraacetic acid for 90 min. They were scanned on a Gilford gel scanner at 360 nm. Results Degradation of chondroitin sulfate by hyaluronidase The alkali-treated chondroitin sulfate used as the substrate for hyaluronidase assay contains large molecular of varying chain size. As shown in Fig. 1, they migrated in the gel as a broad band. By incubating with hyaluronidase the chondroitin sulfate was hydrolyzed to smaller molecules with diminished chain size. They were separated into 2 parts by the modified disc gel electrophoresis. One part which consisted of the smaller chains were concentrated into a thin band and migrated with the buffer front, giving a sharp peak after staining the gel with cetylpyridinium chloride and scanning the gel by the spectrophotometer. The other part was the mixture of the undegraded and the partially
o&---2
3
4
5
LENGTH OF GEL (cm) Fig. 1. Disc gel electrophoresis of chondroitin sulfate. A mixture of 200 fig of chondroitin sulfate and 0.25 NF units of heat inactivated hyaluronidase was run in a gel. After the gel was stained in cetylpyridinium chloride, the whole gel was scanned at 360 nm.
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LENGTH OF GEL b-n) Figs. 2 and 3. Disc gel eleetroghoresis of chondroitin sulfate after partially degradation by hyaluronidase. Chondroitin sulfate (4 me/ml) was incubated with commercial hyaluronidase, 5 NF units/ml. Fifty 1.11of the incubating mixture were drawn at various times, run in a gel and scanned at 360 nm after staining with cetylpyridinium chloride. Fig. 2 shows the result of 20 min incubation, and Fig. 3, SO min. The sharp peaks are the chondroitin sulfate oligosaccharides with low molecular weights concentrated to a single discrete band, and run with the buffer front in the gel electrophoresis.
degraded chondroitin sulfates which migrated as a broad peak behind and separated from the sharp peak. The results of the scanning profiles of the degraded products after incubation for 20 min and 90 min are shown in Fig. 2 and Fig. 3, respectively. The absorbance of the sharp band increases as the time of incubation increases. Fig. 4 shows that the absorbance is linearly proportions to the incubation time; thus the degraded products increase with time of hydrolysis.
0.001 10*
’
20
’
30
’
40
’
50
t
80
’
708090
’
’
MINUTES INCUBATION Fig. 4. Degradation of chondroitin sulfate by hyaluronidase and the duration of incubation. One hundred ~1 containing 600 ~g of chondroitin sulfate in 0.1 mol/l phosphate buffer, pH 5.3, and 0.16 mol/l NaCl was incubated at 37’C with 0.06 NF units of hyaluronidase for different time intervals. The reaction was stopped by heating in a water bath for 5 min. Twenty ~1 of each solution was used for electxophoresis.
281 .8 r
,
5
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15
20
25
30
HYALURONIDASE DEGRADED C~NDROITIN SUt_FATE (pg)
35 NF UNITS OF HYA~URONIDASE (U/ml)
Fig. 5. Linear relationship of the hyaluronidase degraded chondroitin sulfate and absorbance. The hyaluronidase degraded chondroitin sulfate used as a standard for enzyme assay gave a single concentrated band running with the buffer front in the gel electrophoresis. The quantities are linearly related to the absorbance of the peaks. The four peaks shown in the figure represent 13.6 ng, 20.4 tig. 27.2 A% and 34 Pg of the standard chondroitin sulfate, respectively. Fig. 6. Degraded product of chondroitin sulfate versus hyaluronidase concentration. The reaction mixture in a volume of 0.2 ml containing 2 mg ehondroitin suffate, 0.1 moi/i phosphate buffer. pH 5.3, 0.13 moifl NaCI. and different concentration of hyaluronidase was incubated at 37’C for 1 h. The reaction was terminated by heating the reaction mixture in a water bath for 5 min. Twenty pl of each reaction mixture was then assayed for the degraded chondroitin sulfate by gel electrophoresis.
Relationship between the absorbance and the degraded ~h~ndr~~tin sulfate The degraded products obtained by treating the chondroitin sulfate with excess amount of hyaluronidase gave a single sharp band on disc gel electrophoresis. The amount of the degraded chondroitin sulfate is linearly related to the optical density of the peak. This is illustrated in Fig. 5, the degraded chondroitin sulfate between 10 pug and 35 t_lg gave a linear relationship with absorbance. Relationship between hyaluronidase activity and hyaluronidase concentration When the concentration of hyaluronidase was varied, the amount of the degraded chondroitin sulfate produced as determined by the disc gel electrophoresis was linearly proportional to the activity of hyaluronidase. The results are shown in Fig. 6. The detail of the experimental procedure is described in the legend of the figure. From the data of this figure and using the curve in Fig. 5 as a standard, it is calculated that 1 NF unit of hyaluronidase is equivalent to 1216 units of hyaluronidase activity defined in this paper. Comparatiue assays for standard hyaluronidase (NF) by turbidity reducing method, ~o~orimetr~e technique and proposed mi~r~~ssay In the same experimental condition, one NF unit of hyaluronidase from the American Pharmaceutical Association represented 0.35 turbidity reducing unit, 12.2 pg of N-acetylglucosamine demasked by calorimetric technique and 38 pg of chondroitin sulfate degraded by our micro-assay. The experimental condition was at pH 5.3 and 37”C, and in 30 min of reaction time.
282
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1.6
r
0.6
3
4
5
6
7
8
PH Fig. 7. pH optimum of hyaluronidase from different sources. The solid line shows the pH optimum curve for a crude extract of rat testes, the dotted line for the commercial hualuronidase. The buffers used are phosphate buffer (closed circle) and acetate buffer (open circle).
pH optimum
of hyaluronidase
from different
sources
As shown in Fig. 7, the commercially available testicular hyaluronidase showed an optimal reaction at pH 6. A crude extract from rat tested showed an optimal reaction at pH 4.8. While the activities in human serum and urine tested at pH 6 using phosphate buffer or at pH 4.8 using acetate buffer showed a little activity, maximal activity was obtained when the pH of the acetate buffer was adjusted to pH 3.4. The pH at the end of incubation was about pH 4. Similar result was obtained when lactic acid instead of acetic acid was used as a buffer. Preliminary
observation
of hyaluronidase
activity in human serum
Sera were obtained from patients with different diseases, as well as from normal. The level of hyaluronidase activity tested on 26 normal subjects was fairly constant. The average was 892 + 64 units/ml serum. Control data from the disease states are being collected and the results will be reported in a separate paper. Discussion The crux of this method is based on the observation that the degraded products of chondroitin sulfate also precipitate with cetylpyridinium chloride in the gel at an optimal concentration of cetylpyridinium chloride. Furthermore, the degraded products of chondroitin sulfate can be concentrated to a thin single band by the modified disc gel electrophoresis and separated from the intact or partially degraded chondroitin sulfate. In our buffer system, we used borate as a trailing anion. Borate ion has an electrophoretic mobility lower than that of chondroitin sulfate. Thus, in the gel smaller size chondroitin sulfate will be con-
283
tinuously concentrated into a single band and moves in the gel with the buffer front between the leading anion, Cl-, and the trailing anion, borate. While the larger size chondroitin sulfate will be retarded by the gel leaving behind the buffer front and separated according to their size as a broad band shown in Figs. 2 and 3. The chain size of chondroitin sulfate which will not be subjected to the sieving action of 7.5% acrylamide gel is yet to be determined. However, from our previous experiment [ 131, it is estimated to be less than four tetrasaccharide units. In order to increase the sensitivity of the method, one can use lower concentration of acrylamide gel to increase the pore size. Thus, more’of the degraded products will be concentrated in the sharp band. At present hyaluronidase can be determined by turbidimetric, viscosimetric and calorimetric methods. Turbidimetric and viscosimetric methods are unsatisfactory in clinical use [ 121. Only the calorimetric method is widely used by many investigators [ 7,8,20,21]. Even though the micro-assay proposed is slightly cumbersome compared to calorimetric method, we find that our microassay has potential clinical use in assaying biological fluid and tissue. Since a hyaluronidase degraded chondroitin sulfate system is employed, our method would be less susceptible to any interference. Substances which might interfere with the system are nucleic acid and acid glycosaminoglycans. This interference can be prescreened by gel electrophoresis. Furthermore, any sample containing colored substance can be determined with our method accurately. The procedure described has several distinct advantages. First, chondroitin sulfate is easily obtainable in a pure form and in large quantities. Secondly, hyaluronidase from most bacteria can be eliminated [ 161, and thirdly, our sensitivity can be enhanced greatly by using isotope labelled chondroitin sulfate. Two types of hyaluronidase are known to exist in mammalian tissues. They are lysosomal and testicular types. Each type has its distinct pH optimum. Lysosomal hyaluronidase has pH optimum of 3.5, whereas testicular type, 4.5 to 6.0 [ 171. We note that human hyaluronidase has similar pH optimum as of lysosomal origin, as reported by others [ 7,8,19]. Finally, we believe that the proposed new micro-assay for human hyaluronidase using our chondroitin sulfate-gel electrophoresis system can complement the existing simpler calorimetric methods by overcoming interferences. References 1 Mayer, R.L., Kocholaty. W. and Stanton, D. (1948) Proc. Sot. Exptl. Biol. Med. 67. 529-531 2 Meyer. K. and Rapport, M.M. (1952) in Advances in Enzymology. Vol. 13 (Nerd, F.F., ed.), pp. 199236, Interscience Publishers, New York 3 Ginetzinsky. A.G. (1958) Nature 182.1218-1219 4 Berlyne. G.M. (1960) Nature 185, 389-390 5 Knudsen, P.J. and Koefoed, J. (1961) Nature 191,1306-1307 6 Cobbin. L.B. and Dicker, S.E. (1962) J. Physiol. 163.168-174 7 BoUet, A.J., Banner, W.M., Jr. and Nance, J.L. (1963) J. Biol. Chem. 238. 3522-3527 8 Sempinska, E., Keibasinska. E.. Kostka, B. and Mazurowa, A. (1976) Acta Med. Pal. 17. 79-84 9 Pigman, W., Fabianek. J.. Herp, A., Root, L. and &lick. A. (1963) Arthritis Rheum. 6,788 10 Banner, W.M., Jr. and Cantey, E.Y. (1966) Clin. Chim. Acta 13.746-752 11 Salkii. M.L., Hannah, C.L. and McNeil, E.M. (1976) Clin. Biochem. 9.184-187 12 Nothup, S.N., Stasiw. R.O. and Brown. H.D. (1973) Clin. Biochem. 6,220-228 13 Hsu, D.S., Hoffman, P. and Mashburn. T.A.. Jr. (1973) Anal. Biochem. 52. 382-394 14 Tolksdorf, S., M&ready, M.H.. McCullagh, D.R. and Schwenk, E. (1949) J. Lab. Clin. Med. 34. 7489
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15 Bitter, T. and Muir. H.M. (1962) Anal. Biochem. 4.330-334 16 Meyer. K. (1971) in The Enzymes. Vol. 5 (Bayer, P.D.. ed.), pp. 307-320, Academic and London 17 Aronson. N.N.. Jr. and Davidson, E.A. (1967) J. Biol. Chem. 242. 437-444 18 DeSalegui, M. and Pigman, W. (1967) Arch. Biochem. Biophys. 120. 60-67 19 Bowness, J.M. and Tan. Y.H. (1968) Biochim. Biophys. Acta 151,288-290
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