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ENZYME AND TOXIN ASSAYS
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sample in 0.1 ml, and heat in a boiling water bath for 12 min. Cool in a
water bath at room temperature. Shake each tube and read A570within I hr.
Assays Using Labeled Gelatin
Gelatin can be radiolabeled with [3H]acetic anhydride or [3H]formaldehyde, as done for collagen.~4,~5Alternatively, it can be fluorescently labeled
with fluorescein isothiocyanate (FITC).16
Incubate enzyme with radiolabeled gelatin or FITC-gelatin in a volume
of 50 /~1 for an appropriate amount of time at 37°. The reactions are
stopped by adding 100/~1 of 10 mg/ml unlabeled gelatin and 50/.d of 50%
trichloroacetic acid (TCA). Let the reactions stand at 4° for 30 min and
then pellet the protein precipitate by centrifugation in a microcentrifuge.
In the case of radiolabeled gelatin, the supernatant is counted to determine
the counts per minute (cpm). The FITC-labeled peptides in the supernatant
are detected by adding 100 /~1 to 1.0 ml of 0.5 M Tris (pH 8.75) and
measuring the fluorescence using excitation at 490 nm and monitoring
emission at 525 nm. Rather than incubating the labeled gelatin in solution,
it can also be covalently attached to the preactivated surface of a 96-well
plate (Costar, Cambridge, MA), with release of label into the supernatant
being measured.
14 H. Birkedal-Hansen, this series, Vol. 144, p. 140.
15 K. A. Mookhtiar, S. K. Mallya, and H. E. Van Wart, Anal. Biochem. 158, 322 (1986).
16 U. Tisljar and H . - W . Denker, Anal. Biochem. 152, 39 (1986).
[48] A s s a y s for H y a l u r o n i d a s e A c t i v i t y
By WAYNE L. HYNES and JOSEPH J. FERRETTI
Introduction
Hyaluronidases are a group of enzymes [hyaluronate 4-glycanohydrolase (EC 3.2.1.35, hyaluronoglucosamidase), hyaluronate 3-glycanohydrolase (EC 3.2.1.36, hyaluronoglucuronidase), and hyaluronate lyase (EC
4.2.2.1)] that catalyze the breakdown of hyaluronic acid, a mucopeptide
composed of alternating N-acetylglucosamine and glucuronic acid residues. Hyaluronidases are produced by a variety of pathogenic organisms
including group A and C streptococci, pneumococci, staphylococci, and
clostridia. The enzymes are also found in leeches, snake and insect venoms, and malignant tissues. In the pathogens, hyaluronidases are freMETHODS IN ENZYMOLOGY, VOL. 235
Copyright © 1994by AcademicPress, Inc.
All rights of reproduction in any form reserved.
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HYALURONIDASEASSAYS
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quently termed spreading factors because of their ability to break down
the hyaluronic acid component found in the cement substance of host
tissues. A variety of methods have been employed over the years to assay
hyaluronidase activity, but many of the assays appear to be rarely used
today and are discussed only briefly. All methods cited should be applicable to hyaluronidases from either microbial or mammalian sources; however, confirmation of such applicability has not always been done and
may be dependent on the mechanism of hyaluronic acid breakdown. As
there are many different assays for hyaluronidase activity we have separated the methods into groups based on the type of assay performed,
namely, (1) spectrophotometric, (2) radiochemical, (3) fluorogenic, (4)
enzymoimmunological, (5) plate (solid media), (6) chemical, (7) physicochemical, and (8) zymographic analysis.
Spectrophotometric Assay
A sensitive method for the detection of hyaluronidase activity was
developed by Bcnchctrit e t al. ~ based on a shift in maximal absorbance
following interaction of anionic mucopolysaccharides with a carbocyanlne
dye. Hyaluronic acid is frccd from protein contamination by digestion
with pronase, treatment with chloroform-isoamyl alcohol, and precipitation with NaCl-saturated ethanol. The assay as described by Bcnchctdt
e t al. is performed in a final volume of l0/~I. Hyaluronlc acid (l ~g) and
enzyme are incubated at 37° for 60 rain. The reaction is stopped by addition
of 9 ~l of water and freezing. The dye, Stains-all {1-ethyl-2-[3-(1-ethylnaphtho[1,2- d]thiazolin-2-ylidcne) -2-methylpropenyl]naphtho[ 1,2-d]thiazolium bromide; Eastman Organic Chemicals, Rochester, NY} is prcparcd
at 0.1 mM in water containing 50% dioxane (final concentration of dioxane
in the dye solution is 5%), 1 mM acetic acid, and 0.5 mM ascorbic acid.
The dye is added to the reaction mixture to give a final volume of 1.0 ml
and the absorbancy determined at 640 nm against a blank containing dye
(0.9 ml) and water (0.1 ml). One unit of hyaluronldase activity is defined
as the amount of cnzymc required to decrease the absorbance of the
hyaluronic acid-dye complex by 10% after 1 hr incubation at 37°, pH 5.0.
In our hands, this method has been useful for the detection and quantitation of purificd hyaluronldase; however, it is not reliable in the detection
of activity in crude (culture supernatants) preparations where interfering
substances may be present. A modification of the assay carded out in
I L. C. Benchetrit, S. L. Pahuja, E. D. Gray, and R. D. Edstrom, Anal. Biochem. 79,
431 (1977).
608
ENZYME AND TOXIN ASSAYS
[48]
100-/xl volumes has been used by Hotez e t al. 2 to detect hyaluronidase
activity in hookworm larvae. These authors noted that differences between
substrate and products of the reaction were detected only by addition of
glacial acetic acid to the dye. The requirement for the acid may be due
to the different way the Stains-all dye is prepared.
A further modification to this procedure was recently described for
the assay of chondroitin sulfate depolymerase and hyaluronidase activity
in viridans streptococci 2b. In this procedure, the dioxane contains 25 ppm
2,6-di-tert-butyl-4-methylphenol as a stabilizing agent2a,2b; prepared in this
way the dye solution is stable for at least 2 weeks. Assays are performed
in microcentrifuge tubes containing 100/zl of bacterial suspension in 50
mM Tris-C1 buffer pH 7.5,350/xl 0.2 M Na2HPO 4 buffer pH 6.5, and 200
/zl of 2.0 mg/ml hyaluronic acid. Following incubation at 37°, samples (20
/zl) of the reaction mix are removed, mixed with 180/zl of dye solution,
followed by 100/zl of distilled water and the absorbance determined at
620 nm within 30 min of addition of the dye. Removal of the bacterial
cells is not necessary as their presence does not appear to interfere with
development of the hyaluronic acid-dye complex 2b. It was reported in
these studies that a variety of salts (CaC12, FeC13, MgC12, MnSO4, NaCI,
and ZnCI2) at concentrations as low as 1 mM reduce the absorbance of
the hyaluronic acid-dye complex, while addition of EDTA enhances the
absorbance values. 2a Difficulties due to the presence of these salts, in
particular Fe 3÷, can be overcome by routinely including chelators such
as EDTA in the assay. 2a
An alternative colorimetric assay based on the interaction of Alcian
Blue 8GX with hyaluronic acid has been described 3 in which the absorbance of the digest is inversely proportional to the concentration of hyaluronidase. All solutions are prepared in 50 mM sodium acetate buffer,
pH 5.0, containing 50 mM MgCl 2. The hyaluronidase-containing solution
(0.5 ml) to be assayed is added to 200/xl of substrate containing 60/xg
of hyaluronic acid and incubated with shaking at 37° for 25 min. After
incubation, 1 ml of Alcian Blue solution (0.02%) is added, and the tubes are
shaken and immediately centrifuged. The absorbance of the supernatant is
determined at 603 nm.
Recently Pritchard and Lin reported on the use of the thiobarbituric
acid assay, based on the detection of sialic acid, for the assay of hyaluroni2 p. j. Hotez, S. Narasimhan, J. Haggerty, L. Milstone, V. Bhopale, G. A. Schad, and
F. F. Richards, Infect. lmmun. 611, 1018 (1992).
K. A. Homer, L. Denbow, and D. Beighton, Anal. Biochem. 214, in press.
2b K. A. Homer, L. Denbow, R. A. Whiley, and D. Beighton, J. Clin. Microbiol. 31,
1648 (1993).
3 R. H. Pryce-Jones and N. A. Lannigan, J. Pharm. Pharmacol. 31(Suppl.), 92P (1979).
[48]
HYALURONIDASEASSAYS
609
dase activity. 3a The assay is carried out in 200/zl volumes consisting of
100/xl of 2 mg/ml substrate, 20 tzl of 10X buffer (0.5 M ammonium acetate
buffer [pH 6.5], 0.1 M CaC12), and enzyme diluted in 2 mg/ml BSA. The
volume is made up to 200/xl with distilled water. After incubation at 37°
the reaction is terminated by placing the tubes briefly in a boiling water
bath. The tubes are then assayed as described by Skoza and Mohos. 3b To
the reaction tube 0.1 ml of 6% (w/v) thiobarbituric acid, adjusted to pH
9.0 with NaOH, is added to give a final concentration of at least 1%.
Development of the chromophore is by heating the reaction in a boiling
water bath for 7.5 min. An equal volume of dimethyl sulphoxide is added
to intensify the color and the absorbance measured at 549 nm. Development of the color detected in this assay is due to the action of the hyaluronidase breaking the glycosidic bond by elimination (introduction of a double
bond) rather than hydrolysis (addition of water). Acid-hydrolyzed hyaluronic acid does not react in this assay due to the absence of double bonds
in the products 3a.
Radiochemical Assay
The principle of the radiochemical method described by Coulson and
Girken 4 in studies of tissue hyaluronidases is that cetylpyridinium chloride
precipitates hyaluronic acid, but not smaller molecular weight polysaccharides. In this procedure, hyaluronic acid is partially deacylated using
hydrazine and then reacylated in the presence of [3H]acetic anhydride.
The 3H-labeled hyaluronic acid is purified by precipitation, dialysis, and
column chromatography and diluted with cold hyaluronic acid to give a
final concentration of 1.2 mg/ml. Hyaluronic acid (60/.~g in 50/zl of 0.1
M formate buffer, pH 3.5, containing 0.15 M NaCl) is incubated with 50
~l of enzyme solution at 37° for 20 min. Termination of the reaction is
achieved by the addition of 50 ~l of 0.25 M disodium hydrogen phosphate.
To precipitate undigested substrate, 50/~l of a 1% cetylpyridinium chloride
solution is added and the mixture incubated for 30 min at 37°. Following
centrifugation, a 50-~1 aliquot of supernatant is added to l0 ml of scintillation fluid and the radioactivity of the sample determined. Radioactivity
of the blanks, in which the enzyme preparation is added immediately prior
to the disodium hydrogen phosphate (reaction terminator), is subtracted
from the test readings to determine the radioactivity solubilized by the
action of hyaluronidase.
3a D. G. Pritchard and B. Lin, Infect. lmmunol. 61, 3234 (1993).
3b L. Skoza and S. Mohos, Biochem. J. 159, 457 (1976).
4 C. J. Coulson and R. Girkin, Anal. Biochem. 65, 427 (1975).
610
ENZYME AND TOXIN ASSAYS
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An alternative assay which follows the breakdown of 3H-labeled hyaluronic acid is described for the assay of hookworm hyaluronidase. 2 In
this procedure radiolabeled hyaluronic acid is purified following synthesis by human keratinocytes. The labeled substrate is incubated with
the sample and aliquots removed at various time periods and placed into
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
sample buffer. After all samples have been collected, the degraded hyaluronic acid is applied to a polyacrylamide gel. Following electrophoresis, the gel is fixed and prepared for autoradiography. Hyaluronidase
activity is observed as a decrease in the size of the labeled hyaluronic
acid.
Fluorogenic Assay
Hyaluronic acid labeled with the fluorogenic reagent 2-aminopyridine
has been used as the substrate in a rapid, simple, and sensitive assay for
the detection of testicular hyaluronidase) Reactions are set up in 50-fd
volumes containing 1.5 gg of pyridylaminohyaluronate and enzyme in 50
mM sodium acetate buffer containing 0.15 M NaC1. After incubation at
37° for 60 rain, exactly 200/~1 of NaCl-saturated ethanol is added and the
mixture is left at 0° for 30 rain. Following centrifugation, 300/~1 of 0.5 M
sodium acetate buffer, pH 4.0, is added to 100 #1 of supernatant and the
fluorescence of the solution measured at an excitation wavelength of 320
nm and an emission wavelength of 400 nm. The increase in pyridylamino
products is linearly correlated with enzyme concentration under these
conditions.5 The fluorogenic substrate has also been used for determination
of hyaluronidase activity in crude liver extracts, overcoming some of the
problems associated with other assays resulting from the presence of
interfering compounds.
Indirect Enzymoimmunological Assay
Hyaluronectin is a hyaluronic acid-binding proteoglycan which can be
used as a probe in an indirect enzymoimmunological hyaluronidase assay
developed for the detection of small amounts of hyaluronidase in preparations from the leech, bovine testes, hepatoma cell lines, bee venom, and
streptomyces species. 6 Microtiter plates are coated with hyaluronic acid
(100 rag/liter in 0.1 M sodium bicarbonate) and left at 4° overnight prior
5 T. Nakamura, M. Majima, K. Kubo, K. Takagaki, S. Tamura, and M. Endo, Anal.
Biochem. 191, 21 (1990).
6 B. Dclpech, P. Bertrand, and C. Chauzy, J. Immunol. Methods 104, 223 (1987).
[48]
HYALURONIDASE ASSAYS
611
to rinsing with distilled water. Diluted samples are added to wells, in
duplicate, and incubated at 37° for up to 24 hr. After incubation, the
wells are rinsed and incubated with hyaluronectin immune complexes
conjugated with alkaline phosphatase, diluted in 0.1 M sodium phosphate,
pH 7.0, for 4 hr. Alternatively, the procedure can be carried out as a
two-step reaction. Hyaluronectin [150 ng/ml in phosphate-buffered saline
(PBS) supplemented with bovine serum albumin (BSA)] is added to the
wells for 4 hr. After rinsing, the wells are incubated with diluted conjugated
antibodies overnight, whereon the wells are rinsed with PBS and incubated
with substrate (p-nitrophenyl phosphate 1 mg/ml in I M diethanolamine
with 1 mM MgCI 2 at pH 9.8) at 37° for 1-2 hr. Hyaluronidase activity is
indicated by a decrease in absorbance, measured at 405 nm.
Plate (Solid Media) Assays
A number of approaches have been developed for the assay of hyaluronidase using solid media. One of the simplest plate assays is that
described by Smith and Willett 7to screen bacteria for the ability to produce
hyaluronidase. One hundred milliliters of brain-heart infusion broth containing 1% agar is sterilized by autoclaving and allowed to cool to 46 °.
An aqueous 2 mg/ml solution of sterilized hyaluronic acid is added to a
final concentration of 400/~g/ml. A 5% filter-sterilized solution of bovine
albumin fraction V is added with constant stirring to give a final concentration of 1% in the medium. The agar mix is poured into petri dishes, allowed
to solidify, and stored at 4 ° until required. Bacteria are inoculated either
onto the surface or as stabs into the media prior to incubation at 37° .
Following overnight incubation, the plates are flooded with 2 N acetic
acid and allowed to stand for 10 rain. Hyaluronidase activity is detected
as a zone of clearing around the bacterial colonies/stab in a cloudy background, resulting from acetic acid precipitation of an albumin-nondegraded hyaluronic acid complex.
This assay medium can also be used to test liquid samples, such as
bacterial culture supernatants and purified or partially purified preparations, for hyaluronidase activity by addition of sodium azide (0.1%,w/v)
to the medium prior to pouring into petri dishes. Wells are cut into the
solidified assay medium and filled with the sample to be assayed. After
incubation, the undigested hyaluronic acid/albumin complex is precipitated with acetic acid. Zones of clearing around wells indicate hyaluronidase activity. 8
7R. F. Smith and N. P. Willett, Appl. Microbiol. 16, 1434(1968).
s W. L. Hynes and J. J. Ferretti, Infect. lmmun. 57, 533 (1989).
612
ENZYME AND TOXIN ASSAYS
[48]
Another plate assay is based on the precipitation of undigested hyaluronic acid by cetylpyridinium chloride.9 Assay plates are prepared consisting
of 1 mg/ml hyaluronic acid in 1.5% (w/v) agarose buffered with 50 mM
sodium citrate (pH 5.3) and containing 0.02% sodium azide (buffer A).
Equal volumes of stock solutions of 3% agarose and 2 mg/ml hyaluronic
acid in buffer A are mixed at 60° with constant stirring for about 1 min
to ensure complete mixing prior to pouring into petri dishes. When the
medium solidifies, holes are punched into the medium, filled with the
sample to be assayed, and incubated at 37° for 18-20 hr. After incubation,
the gel is flooded with 10% (w/v) cetylpyridinium chloride in water and
examined against a dark background for zones of clearing around the
wells. Zone diameters of known standards can be measured and plotted
against units of enzyme using a semilogarithmic scale, thereby allowing an
estimation of the amount of hyaluronidase activity in a particular sample.
We have used both of the plate assays in our laboratory for detection
of streptococcal hyaluronidase 8'1° and found both to be suitable for assay
of liquid preparations. The Smith and Willett assay plates 7 have the added
advantage of being able to detect production of hyaluronidase by bacterial
colonies. A modification of the cetylpyridinium chloride precipitation
method was described by Balke and Weiss, H who used it to detect production of hyaluronidase during growth of different anaerobic clostridial
strains.
Chemical Assays
Liberation of Reducing Sugars
Assay of hyaluronidase by liberation of reducing sugars is based on
the hydrolysis of hyaluronic acid into its reducing sugar components.
Results are calculated as a percentage of the total reducing sugar and
expressed as equivalents of glucose. 12 The measurement of a decrease
in viscosity of hyaluronic acid-containing solution as determined in the
viscosity reduction method is a rapid reaction (depolymerization), whereas
the hydrolysis of hyaluronic acid with liberation of reducing sugars is a
slower reaction. 12 Based on the reduction of ferricyanide to ferrocyanide
9 p. G. Richman and H. Baer, Anal. Biochem. 109, 376 (1980).
10 W. L. Hynes and J. J. Ferretti, in "Streptococcal Genetics" (J. J. Ferretti and R. Curtiss
III, eds.), p. 150. American Society for Microbiology, Washington, D.C., 1988.
11 E. Balke and R. Weiss, Zentralbl. Bakteriol. Mikrobiol. Hyg., Ser. A 257, 317 (1984).
12 K. Meyer, E. Chaffee, G. L. Hobby, and M. H. Dawson, J. Exp. Med. 73, 309 (1941).
[48]
HYALURONIDASE ASSAYS
613
a "colorimetric" assay was developed and used to assay bacterial hyaluronidases. 13
Liberation of Acetylamino Sugars
As discussed by Meyer, 14 early procedures used for the detection
of acetylglucosamine 12,15 could not be used to follow the hydrolysis of
hyaluronic acid. The problem was associated with the release of acetylglucosamine in excess of the weight of polysaccharide added to the initial
reaction mix.
Reissig et al.16 optimized a colorimetric method for the estimation of
acetylamino sugars. Later, Rouleau 17 described the effects of divalent
cations, cryoprotective agents, and sulfhydryl compounds on the colorimetric assay for acetylglucosamine. The interfering effects reported in
the detection of the acetyl sugar emphasized a need for caution in interpretation of results in such assays, as Ca z÷ actually had an activating effect
on bull sperm hyaluronidase. Methods based on those of Reissig et al. 16
have been used to study hyaluronidase activity from Propionibacterium
acnes 18 and Streptococcus dysgalactiae. 19 Hamai 19 et al. modified the
method of Ingham et al. 18 for assay using small volumes. To 50/zl of a
0.2% sodium hyaluronate aqueous solution is added 50/xl of 0.1 M phosphate buffer, pH 6.2, containing 0.02% BSA and 20/zl of buffer containing
enzyme. After incubation at 37° for 10 min, the reaction mixture is boiled
for 2 min and the increase in reducing sugars assayed using N-acetylglucosamine as a standard.
Absorption o f Unsaturated Uronides
Production of unsaturated uronides results in an increase in absorption
of UV light. 13'2°'21 An increase in the UV absorption (230-235 nm) of a
hyaluronic acid solution, following incubation with hyaluronidase, suggests elimination rather than hydrolysis of the hyaluronic acid. 21
13 A. Linker, this series, Vol. 8, p. 650.
14 K. Meyer, Physiol. Rev. 27, 335 (1947).
15 j. H. Humphrey, Biochem. J. 40, 442 (1946).
t6 j. L. Reissig, J. L. Strominger, and L. F. Leloir, J. Biol. Chem. 217, 959 (1955).
17 M. Rouleau, Anal. Biochem. 103, 144 (1980).
is E. Ingham, K. T. Holland, G. Gowland, and W. J. Cunliffe, J. Gen. Microbiol. 115,
411 (1979).
19 A. Hamai, K. Morikawa, K. Horie, and K. Tokuyasu, Agric. Biol. Chem. 53, 2163 (1989).
2o H. Greiling, H. W. Stuhlsatz, and T. Eberhard, Hoppe-Seyler's Z. Physiol. Chem. 340,
243 (1965).
21 T. Ohya and Y. Kaneko, Biochim. Biophys. Acta 198, 607 (1970).
614
ENZYME AND TOXIN ASSAYS
[48]
Physicochemical Assay
Mucin Clot Prevention
The mucin clot prevention (MCP) assay is based on the coprecipitation
of native hyaluronic acid with protein to form a mucin clot. When hyaluronic acid is digested with hyaluronidase, the quality and character of the
clot is reduced. The MCP test originally described by Robertson et al. 22
was later modified by McClean 23and it is this modification which has been
used most widely. Meyer TM discussed the limitations and some of the
problems associated with the MCP test. A modification of the MCP test
was described by Unsworth for the detection of hyaluronidase production
by Streptococcus rnilleri. 24 Broth culture supernatants (25/~1) are double
diluted in 25 ~1 volumes in a microtiter plate using cold distilled water.
After dilution, 25 ~1 of water is added to each well, for a final volume of
50/zl, followed by 50/~1 of hyaluronic acid prepared as follows: 8 ml of
cold diluted india ink (20 ml distilled water to 0.01 ml of india ink) is added
to Bacto-AHT substrate according to the manufacturer's instructions except for the addition of india ink. The microtiter tray is shaken, incubated
at 37° for 20 min, then cooled at 4 ° for 30 min prior to addition of 25/zl
of cold acetic acid to each well. The tray is shaken to ensure mixing of
all components. The absence of a black clot indicates hyaluronidase activity, with the titer of hyaluronidase being the highest dilution that fails to
show clotting. A modification of the MCP test using india ink to follow
clot development has been utilized in the detection of hyaluronidase antibody in patients following streptococcal infection. 25'26
Viscosity Reduction
The viscosity reduction method measures the rate of decrease in viscosity of a solution of hyaluronic acid. Meyer TMdescribed a standardized
procedure to overcome some of the many variations in this assay (substrate
and sodium chloride concentration, pH, and temperature) which occurred
between different laboratories. Tirunarayanan and Lundblad 27 found the
viscosimetric method to be a reliable assay in investigations into the
hyaluronidase produced by Staphylococcus aureus.
22 W. Robertson, M. W. Ropes, and W. Bauer, J. Biol. Chem. 133, 261 (1940).
23 D. McClean, Biochem. J. 37, 169 (1943),
24 p. F. Unsworth, J. Clin. Pathol. 42, 506 (1989).
25 S. A. Halperin, P. Ferrieri, E. D. Gray, E. L. Kaplan, and L. W. Wannamaker, J. Infect.
Dis. 155, 253 (1987).
26 R. A. Murphy, Appl. Microbiol. 23, 1170 (1972).
27 M. O. Tirunarayanan and G. Lundblad, Acta Pathol. Microbiol. Scand. 73, 211 (1968).
[48]
HYALURONIDASEASSAYS
615
Turbidimetric A s s a y
The turbidimetric a s s a y is b a s e d on the observation that acidified hyaluronic acid in the p r e s e n c e of dilute serum forms a stable colloidal suspension. Once depolymerized, the hyaluronic a c i d - s e r u m mixture remains
clear.14,28-31 U s e o f a modified turbimetric assay allowed O h y a and K a n e k o
to describe a hyaluronidase f r o m S t r e p t o m y c e s hyalurolyticus nov. sp. zl
A semiquantitative m i c r o a s s a y b a s e d on the turbidimetric assay is reported to be sensitive, simple, reproducible, and economical. 32
Zymographic Analysis
Z y m o g r a p h y , a p r o c e d u r e allowing visualization of e n z y m e activity
following electrophoretic fractionation, has been used for the qualitative
analysis of a n u m b e r of hyaluronidases. 2'33-38 Z y m o g r a p h i c analysis can
be carried out on a variety of solid supports such as agar, 33 acrylamide, 34'35'37'38 or cellulose acetate m e m b r a n e s . 36
A b r a m s o n and F r i e d m a n 33 used a modification of the MCP a s s a y to
detect hyaluronidase activity in concentrated preparations f r o m S t a p h y l o c o c c u s a u r e u s and S t r e p t o c o c c u s p y o g e n e s as well as samples oftesticular
hyaluronidase. Following electrophoresis in 1% N o b l e agar with barbital
buffer, p H 8.6, on a glass slide, hyaluronic a c i d - h o r s e s e r u m - a g a r is
layered onto the slide. After incubation, the slides are treated with acetic
acid and examined for a clear area against a background of precipitated
hyaluronic acid substrate.
An alternative to electrophoresis in agar has b e e n described by H e r d
et al. 36 in which electrophoresis is carried out on cellulose acetate m e m branes. After electrophoresis, the m e m b r a n e is overlaid with a second
m e m b r a n e saturated with hyaluronic acid, and the sandwich is incubated
at 37 ° for 30 min. Following incubation, the overlay m e m b r a n e is stained
with alcian blue, w a s h e d and allowed to dry. Hyaluronidase activity is
seen as white b a n d s in a blue background.
28M. B. Mathews, this series, Vol. 8, p. 654.
z9j. Komender, A. Golaszewska, and H. Malczewska, Histochemie 35, 219 (1973).
30A. Dorfman and M. L. Ott, J. Biol. Chem. 172, 367 (1948).
31 S. Tolksdorf, M. H. McCready, D. R. McCullagh, and E. Schwenk, J. Lab. Clin. Med.
34, 74 (1949).
32A. N. Ibrahim and M. M. Streitfeld, Anal. Biochem. 56, 428 (1973).
33C. Abramson and H. Friedman, Proc. Soc. Exp. Biol. Med. 125, 256 (1967).
34B. Fiszer-Szafarz, Anal. Biochem. 143, 76 (1984).
35B. Fiszer-Szafarz and E. De Maeyer, Somatic Cell Mol. Genet. 15, 79 (1989).
36j. K. Herd, J. Tschida, and L. Motycka, Anal. Biochem. 61, 133 (1974).
37Von M. Lietlander and H. Stegemann, Hoppe-Seyler's Z. Physiol. Chem. 349, 157 (1968).
38B. Steiner and D. Cruce, Anal. Biochem. 200, 405 (1992).
616
ENZYME AND TOXIN ASSAYS
[48]
F i s z e r - S z a f a r z 34'35 incorporated hyaluronic acid into polyacrylamide
gels prior to electrophoresis. Following electrophoresis, the gel is washed
with water and buffer (50 mM acetic acid, 50 mM KH2PO4,0.15 M NaCI,
2.7 mM sodium EDTA, pH 6.0) before incubation at 37° for 16 h to allow
the enzyme to degrade the hyaluronic acid included in the gel. The gel is
washed with water and soaked in 50% aqueous formamide before staining
with Stains-all. A stock solution of 1 mg/ml Stains-all in formamide is
diluted 20-fold with 9 volumes of formamide and 10 volumes of water.
Staining is carried out in the dark for 2 days before gels are washed in
water. Hyaluronidase activity is indicated by pink bands (polyacrylamide
staining) in a blue background (undegraded hyaluronic acid staining). Utilizing this procedure, polymorphism among various hyaluronidases has
been noted, 34'35 including those from a group A streptococcus and from
Streptomyces hyalurolyticus. Stains-all sensitivity is considered to be an
advantage for the detection of smaller amounts of hyaluronidase in comparison with dyes such as alcian blue or toluidine blue. 34'37 Hotez et al. 2
modified this procedure to incorporate SDS into the gel allowing for separation of enzymatic activity as a function of molecular weight. Following
electrophoresis at low voltage, the gel is washed with Triton X-100 to
displace the SDS, then washed with buffer before staining.
A zymographic analysis of hyaluronidase activity from Propionibacterium acnes, Streptococcus pyogenes, Staphylococcus aureus, and Treponema pallidurn has been reported. 38 Electrophoresis is performed using
native polyacrylamide gels followed by overlaying the gels with a 1%
agarose zymogen containing (0.8 mg/ml) hyaluronic acid and 1% BSA in
0.3 M sodium phosphate buffer, pH 5.3. The substrate zymograph can be
prepared ahead of time and stored at 4 ° until required. Polyacrylamide
gels to be assayed for hyaluronidase activity are washed with buffer (0.3 M
sodium phosphate buffer, pH 5.3) and placed directly onto the substrate
agarose zymograph. After incubation at 37° for up to 2 days, the polyacrylamide gel is removed and the agarose replica developed by immersion in
2 M acetic acid. Areas ofhyaluronic acid breakdown, indicating hyaluronidase activity, appear as clear zones in an opaque background. The authors
compared their results to those of Fiszer-Szafarz 34 and reported that agarose replica gels are as sensitive as incorporation of substrate into the
acrylamide and had the advantage that the gels were much easier to handle.
Additionally, the replicas did not have to be stored in the dark and did
not fade with age, apparently being stable indefinitely when stored in
acetic acid or water.