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384 Nawy et al.

activity can then be expressed in absolute units. This rapid and sensitive technique,

1000 times more sensitive than the commonly used assays, facilitates evaluation of

hyaluronidase in most biological samples including the conditioned media of cultured

cells (see Note 1). The present assay facilitated the isolation and sequence analysis of

the first vertebrate somatic hyaluronidase, human plasma hyaluronidase, Hyal-1 (14).

This permitted detection of six paralogous hyaluronidase sequences in the human

genome (15, see Note 2).

By using a standard amount of a defined hyaluronidase preparation, the proce-

dure is easily converted to a rapid assay for hyaluronidase inhibitors. The latter,

though first described in 1946, are a class of molecule about which very little is known

(16–18). They are obviously important in the intricate modulation of HA deposition.

2. Materials

1. Human umbilical cord HA (ICN, Costa Mesa, CA), morpholineethane sulfate

(MES), biotin, sulfo-NHS, DMSO stock solution, stock solution of 100 mM 1-ethyl-

3-(3-dimethylaminopropyl) carbidodiimide (EDC), and 4 M guanidine-HCl.

2. Bovine testicular hyaluronidase (Wydase〉, Wyeth-Ayerst Co., Philadelphia, PA).

Covalink-NH plates, and PBS containing 2 M NaCl and 50 mM MgSO

(washing buffer).

3. COVALINK-NH microtiter plates (NUNC, Placerville, NJ), buffer: 0.1 M formate, pH

3.7, 0.1 M NaCl, 1% Triton X-100, 5 mM saccharolactone for lysosomal acid-active

hyaluronidases, and 10.5 mL of PBS containing 0.1% Tween 20.

4. 96-well vinyl assay plates (Costar, Cambridge, MA).

5. N-hydroxysulfosuccinimide (Sulfo-NHS) and biotin hydrazide (Pierce, Rockford, IL).

6. Dimethyl sulfoxide (DMSO) and guanidine hydrochloride (Fisher Scientific,

Pittsburg, PA).

7. O-Phenylenediamine (OPD, Calbiochem, La Jolla, CA).

8. Avidin-biotin complex (ABC kit, Vector Labs, Burlingame, CA).

9. All other reagents are from Sigma Chemical Company (St. Louis, MO).

3. Methods

3.1. Preparation of Biotinylated HA (bHA)

1. Dissolve 100 mg of human umbilical cord HA in 0.1 M morpholineethane sulfate (Mes),

pH 5.0, to a final concentration of 1 mg/mL, and allow to dissolve for at least 24 h at 4°C

prior to the coupling with biotin.

2. Add sulfo-NHS to the HA-Mes solution to a final concentration of 0.184 mg/mL.

3. Dissolve biotin hydrazide in DMSO as a stock solution of 100 mM and add to the HA

solution to a final concentration of 1 mM.

4. Prepare a stock solution of 100 mM 1-ethyl-3-(3-dimethylaminopropyl) carbidodiimide

(EDC) in dH

O and add to the HA-biotin solution to a final concentration of 30 µM. Stir

overnight at 4°C.

5. Remove the unlinked biotin and EDC by the addition of 4 M guanidine-HCl and dialysing

against 1000× volumes of dH

O with at least three changes. The dialyzed bHA can be

aliquoted and stored at –20°C for up to several months.

3.2. Immobilization of bHA onto Wells of Microtiter Plates

1. Dilute sulfo-NHS to 0.184 mg/mL in dH

O with the bHA at a concentration of 0.2 mg/mL

and pipet into 96-well Covalink-NH plates at 50 µL per well.

Hyaluronidase Activity and Inhibitor 385

2. Dilute EDC to 0.123 mg/mL in dH

O and pipet into the Covalink plates with the HA solu-

tion yielding a final concentration of 10 µg/well HA and 6.15 µg/well EDC (see Fig.1).

3. Incubate the plates either overnight at 4°C or for 2 h at 23°C. Remove coupling solution

by shaking and washing the plates three times in PBS containing 2 M NaCl and 50 mM

MgSO

(washing buffer). The plates can be stored at 4°C for up to 1 wk in this buffer.

3.3. Assay for Hyaluronidase Activity

1. Equilibrate the plates with 100 µL/well assay buffer: 0.1 M formate, pH 3.7, 0.1 M NaCl,

1% Triton X-100, 5 mM saccharolactone for lysosomal acid-active hyaluronidases (see

Note 3). Substitute 0.1 M formate, pH 4.5, for the PH-20 enzyme, and a neutral buffer for

neutral-active enzymes.

2. Prepare a set of standards for the calibration of enzymatic activity against relative turbidity

reducing units (rTRUs) by serial dilutions of Wydase or other hyaluronidase with a known

activity, in the appropriate buffer, from 1.0 to 1 × 10

–6

rTRU and assay 100 µL/well, the

standard enzyme reaction volume, in triplicate (see Note 4).

3. Pipet unknown samples into plate, 100 µL/well, in triplicate. Preliminary experiments must

be performed to determine the proper dilution of the enzyme preparation. In order to estab-

lish a valid unit of enzyme activity, the range in which activity is proportional to levels of

enzyme must be established (see Figs. 2 and 3). Samples from sources possessing very

low activity may be enhanced by a preliminary immunoaffinity-purification step.

4. Incubate samples in plates for 30–60 min at 37°C.

5. Include positive and negative control wells (no enzyme or no avidin-biotin complex), in

triplicate.

Fig. 1. Structure of the biotinylated disaccharide repeating unit of HA resulting from a

reaction between HA, biotin-hydrazide, and EDAC (Reprinted by courtesy of Academic Press

from ref. 14).

386 Nawy et al.

6. Terminate the reaction by addition of 100 µL/well of 6 M guanidine-HCl, followed by

three washes using the washing buffer.

7. Prepare ABC in 10.5 mL of PBS containing 0.1% Tween 20 (preincubated for 30 min at

room temperature during the hyaluronidase incubation).

8. Add 100 µL/well ABC solution, and incubate for 30 min at room temperature. Do not add

the ABC solution to the negative control wells.

9. Wash the plate five times with the washing buffer, and add 100 µL/well of an OPD

substrate by dissolving one 10-mg tablet of OPD in 10.5 mL of 0.1 M citrate-phosphate

buffer, pH 5.3, plus 7 µL of 30% H

10. Incubate the plate in the dark for 5–10 min and read using a 492-nm filter in an ELISA

plate reader (Titertek Multiskan PLUS, ICN) and monitor by computer using the Delta

Soft II plate reader software from Biometallics (Princeton, NJ).

11. A standard curve is generated by a four-parameter curve-fit of the serial dilutions of the

hyaluronidase standard and unknowns.

3.4. General Considerations

1. Since the activity of hyaluronidases can be unstable, particularly in the absence of deter-

gents, samples should be kept on ice. Stability on freeze-thaw of any unknown enzyme

preparation should be determined ahead of time, before large-scale experiments are under-

taken and frozen.

2. Similarly, a variety of buffers should be compared, to establish the optimal buffer for a

particular system. Acetate buffer may inhibit activity, compared to a formate buffer at the

same pH, as is observed in embryonic chick brain extracts (19). Inclusion of low concentra-

tions of reducing agents (1–5 mM dithiothreitol) should also be tested for their effect on

activity. This is particularly important at low protein concentrations, less that 0.05 mg/mL.

3. Hyaluronidase inhibitors appear to be ubiquitous, and may interfere with detection of

hyaluronidase activities, particularly in preparations that are crude extracts. Occasion-

ally, extracts must be diluted out. The inhibitors appear to occur at more limiting concen-

trations than enzyme. Paradoxically, greater levels of apparent hyaluronidase activity are

observed in plasma samples that are more diluted.

Fig.2. A four-parameter curve-fit of bovine testicular hyaluronidase assays as standard reac-

tions performed at pH 3.7, diluted from 1.0 to 1 × 10

–6

rTRU/well. (Reprinted by courtesey of

Academic Press from ref. 14.)

Hyaluronidase Activity and Inhibitor 387

4. Some hyaluronidase inhibitors are Mg

-dependent (20). For this reason, prior optimization

of assay conditions is necessary for each new system. The effect of EDTA (1–10 mM,

neutral pH) should be examined ahead of time. This may also explain why occasionally

differences can be observed, in assays of hyaluronidase as well as of its inhibitors, between

PBS (phosphate-buffered saline) and PBS-CMF (PBS calcium- and magnesium-free), or

between serum and plasma that has been collected with EDTA or citrate.

5. For detecting low levels of activity, such as in cell culture media, sensitivity of the assay

can be enhanced by increasing the incubation time from 1 h up to 12 h. This is useful for

detecting low levels of activity. However, to maintain a valid unit of enzyme activity, time

dependency must be demonstrated.

6. Any enzyme assay is time-dependent. The use of multichannel pipettes facilitates rapid

addition of reagents and enzyme to the microtiter plates, ensuring reproducibility and stan-

dardizes the reaction time. The assay procedure can be further optimized by initially placing

both the enzyme standards and the unknowns into a vinyl assay plate (Costar) in the same

amounts and arrangement intended for the substrate-coated reaction plate (Covalink-NH).

Once samples are loaded into the vinyl template plate, they can be transferred into the

substrate-coated reaction plates with great speed and efficiency.

Fig.3. (A) Linearity of the enzyme reation over a 30-min incubation period, comparing three

different hyaluronidases. 0.01 rTRU/well was utilized at each time and assayed in triplicate.

(B) Kinetic analyis of log dilutions of immunoaffinity-purified recombinant human plasma

hyaluronidase (Hyal-1). The enzyme, from 1.0 to 0.001 rTRU/well, was assayed from 0 to 30 min.

388 Nawy et al.

7. Kinetic analysis of data can be attempted. However, the HA substrate in this assay is

bound to the microtiter plate, and solid–liquid interface interactions differ significantly

from the true solution chemistry assumed by Michaelis-Menten analysis.

4. Notes

1. An inherited disorder involving absence of serum hyaluronidase activity was described

recently (21,22). The present assay can easily be established as a routine clinical labora-

tory procedure and, requiring only 1-µL samples of serum for each determination, can be

used for genetic screening.

2. A paralogous family of hyaluronidase-like sequences have been identified (15), three

each at chromsomes 3p21.3 (HYAL1, 2, and 3) and 7q31.3 (HYAL4, SPAM1, the gene

for the sperm-specific activity, PH-20, and HYALP1, a pseudogene). The product of

HYAL2, Hyal-2, is an unusual hyaluronidase activity, degrading high-molecular-weight

HA only to an intermediate 20-kDa-sized product. The present assay does not detect Hyal-2

activity, since the 20-kDa HA oligosaccharide would remain bound to the mirotiter plate.

For the assay of Hyal-2 activity, the colorimetric assay of Reissig et al. is recommended

(23). That assay is a measure of the new reducing N-acetylglucosamine terminus gener-

ated by each cleavage reaction. On the other hand, leech hyaluronidase, which is an endo-

β-glucuronidase rather than an endo-β-N-acetylglucosaminidase, is not detected by

the Reissig assay, but can be assayed using the present procedure.

3. The saccharolactone (

D-saccharic acid-1,4-lactone, Sigma) is an inhibitor of β-glucuronidase

activity (24). The enzyme catalyzes a reaction that produces false positives in the Reissig

assay, generating reducing terminal N-acetylglucosamine residues. The importance of this

in the microtiter assay has not been established. It may depend on the activity of β-glucu-

ronidase in the particular extract being examined.

4. The definition of one relative turbidity reducing unit (rTRU) is defined as the amount of

enzyme that reduces the turbidity producing capacity of 0.2 mg HA to 0.1 mg in 30 min at

37°C (25).

Acknowledgments

This work was supported by National Institutes of Health (USA) Grant 1P50 DE/

CA11912, to R. S., and by Lion Corporation, Kanagawa, Japan, to K. M.

References

1. Kreil, G. (1995) Hyaluronidases—a group of neglected enzymes. Protein Sci. 4, 1666–1669.

2. Frost, G. I., Csóka, T. and Stern, R. (1996) The Hyaluronidases: a chemical, biological

and clinical overview. Trends Glycosci. Glycotechnol. 8, 419–434.

3. Csóka, T. B., Frost, G. I., and Stern, R. (1997) Hyaluronidases in tissue invasion. Invasion

Metastasis, 17, 297–311.

4. Laurent, T. C. and Fraser, J. R. (1992) Hyaluronan. Faseb J. 6, 2397–2404.

5. Laurent, T. C. (ed.), The Chemistry, Biology and Medical Applications of Hyaluronan and

its Derivatives, Portland Press, London, UK, 1998.

6. Toole, B. P. (1991) Proteoglycans and hyaluronan in morphogenesis and differentiation,

in Cell Biology of the Extracellular Matrix (Hay, E. D., ed.), Plenum Press, New York,

NY, pp. 259–294.

Hyaluronidase Activity and Inhibitor 389

7. Knudson, W., Biswas, C., Li, X. Q., Nemec, R. E., and Toole, B. P. (1989) The role and

regulation of tumor-associtated hyaluronan, in The Biology of Hyaluronan (Evered, D.,

and Whelan, J., eds.), Wiley, Chichester, UK, pp. 150–169.

8. Weigel, P. H., Fuller, G. M., and LeBoeuf, R. D. (1986) A model for the role of hyaluronic

acid and fibrin in the early events during the inflammatory response and wound healing.

J. Theor. Biol. 119, 219–234.

9. Dorfman, A., Ott, M. L., and Whitney, R. (1948) The hyaluronidase inhibitor of human

blood. J. Biol. Chem. 174, 621–629.

10. Benchetrit, L. C., Pahuja, S. L., Gray, E. D., and Edstrom, R. D. (1977) A sensitive method

of the assay of hyaluronidase activity. Anal. Biochem. 79, 431–437.

11. De Salegui, M., Plonska, H., and Pigman, W. (1967) A comparison of serum and testicular

hyaluronidase. Arch. Biochem. Biophys. 121, 548–554.

12. Stern, M. and Stern, R. (1992) An ELISA-like assay for hyaluronidase and hyaluronidase-

inhibitors. Matrix 12, 397–403.

13. Frost, G. I. and Stern, R. (1997) A microtiter-based assay for hyaluronidase activity not

requiring specialized reagents. Anal. Biochem. 251, 263–269.

14. Frost, G. I., Csóka, T. B., Wong, T., and Stern, R. (1997) Purification, cloning, and expres-

sion of human plasma hyaluronidase. Biochem. Biophys. Res. Commun. 236,10–15.

15. Csóka, A. B., Scherer, S. W., and Stern, R. (1999) Expression analysis of six paralogous

human hyaluronidase genes clustered on chromosomes 3p21 and 7q31. Genomics 60,

356–361.

16. Haas, E. (1946) On the mechanism of invasion. I. Antinvasin I, An enzyme in plasma.

J. Biol. Chem. 163, 63–88.

17. Dorfman, A., Ott, M. L., and Whitney, R. (1948) The hyaluronidase inhibitor of human

blood. J. Biol. Chem., 223, 621–629.

18. Moore, D. H. and Harris, T. N. (1949) Occurrence of hyaluronidase inhibitors in fractions

of electrophoretically separated serum. J. Biol. Chem. 179, 377–381.

19. Polansky, J. R., Toole, B. P., and Gross, J. (1974) Brain hyaluronidase: changes in activity

during chick development. Science 183, 862–864.

20. Mathews, M. B., Moses, F. E., Hart, W., and Dorfman, A. (1952) Effects of metals on the

hyaluronidase inhibitor of human serum. Arch. Biochem. Biophys. 35, 93–100.

21. Natowicz, M. R., Short, M. P., Wang, Y., Dickersin, G. R., Gebhardt, M.C., Rosenthal, D.

I., Sims, K. B., and Rosenberg, A. E.(1996) Clinical and biochemical manifestations of

hyaluronidase deficiency. N. Engl. J. Med. 335, 1029–1033.

22. Triggs-Raine, B., Salo, T. J., Zhang, H., Wicklow, B. A., and Natowicz, M. R. (1999)

Mutations in HYAL1, a member of a tandemly distributed multigene family encoding

disparate hyaluronidase activities, cause a newly described lysozomal disorder,

mucopolysaccharidosis IX. Proc. Natl. Acad. Sci. (USA) 96, 6296–6300.

23. Reissig, J. L., Strominger, J. L., and Leloir, L. R. (1955) J. Biol. Chem. 217, 298–305.

24. Orkin, R.W. and Toole, B. P. (1980) Isolation and characterization of hyaluronidase

from cultures of chick embryo skin- and muscle-derived fibroblasts. J. Biol Chem. 255,

1036–1042.

25. Tolksdorf, S., McCready, M. H., McCullagh, D. R., and Schwenk, E. (1949) The

turbidometric assay of hyaluronidase. J. Lab. Clin. Med. 34, 74–79.

Substrate Gel and Inverse Substrate Gel Technique 391

391

From:

Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols

Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

Detecting Hyaluronidase and Hyaluronidase Inhibitors

Hyaluronan-Substrate Gel and -Inverse Substrate Gel Techniques

Kazuhiro Mio, Antonei B. Csóka, Susan Stair Nawy,

and Robert Stern

1. Introduction

Hyaluronidases are a group of enzymes that degrade the glycosaminoglycan

hyaluronan (HA, hyaluronic acid). Many types of hyaluronidases are reported, from

prokaryotes to eukaryotes (1,2). These enzymes have a wide variety of properties,

including substrate specificity, inhibitor sensitivity, and a range of pH optima. Strep-

tomyces hyaluronidase, and the venom hyaluronidases from bee, snake, and scorpion

are active at neutral pH. Hyal-1, the best-characterized somatic hyaluronidase (3–5),

product of one of the six hyaluronidase-like sequences in the human genome (6), is an

acid-active enzyme with an optimum at pH 3.7. The sperm-specific PH-20 (7) has

apparently two pH optima, pH 4.5 and 7.5, resulting possibly from two forms of the

enzyme, membrane-bound and soluble (8,9).

When analysis is being attempted for the first time of the hyaluronidase activity in

a particular biological source, several problems are encountered. The pH optimum of

the activity is not known. In addition to the very low levels of hyaluronidase in most

tissues, there are hyaluronidase inhibitors. These appear to be ubiquitous, and are

most apt to contaminate hyaluronidase preparations in the crude state, particularly in

the early steps of an enzyme purification procedure. Little is known about these

inhibitors, a class of molecule first described in 1946 (10–12). However, there has

been little progress in the isolation and characterization of this potentially important

class of molecules.

Two types of gel procedures are described here that manage to overcome some of

these problems. The HA-substrate gel procedure was developed specifically for the

detection of hyaluronidase activity in crude extracts and in other complex mixtures

(13,14). This procedure can be run using a wide range of pH values and in various

392 Mio et al.

buffers. The procedure also separates enzyme from inhibitor and permits detec-

tion of hyaluronidase activity in the presence of inhibitors. The inverse HA-sub-

strate gel procedure was developed to examine precisely these hyaluronidase

inhibitors (15).

The HA-substrate gel procedure requires addition of HA into the gel mixture before

polymerization. The gel is stained with Alcian blue. Hyaluronidase activity appears

as a white clearing on a light blue background. Double staining with Coomassie blue

enhances the sensitivity of the procedure. Proteins that remain in the gel, particularly

HA-binding proteins, appear as dark blue bands. These are eliminated by interposing

a pronase digestion step. Some proteins, when present at a particularly high concen-

tration, can prevent penetration of dye into the gel, thus causing a false positive.

However, these are also eliminated by the pronase digeston step.

The two techniques can provide much useful information on the nature of hyalu-

ronidase activities and their inhibitors: pH optima, ion and co-factor requirements,

and their relative molecular sizes, even in the crudest extracts.

2. Materials

2.1. HA-Containing Sodium Dodecyl Sulfate (SDS) Gels

1. Human umbilical cord HA (ICN Biomedicals, Costa Mesa, CA). For the stock solution,

dissolve 100 mg of HA in 100 mL of H

O (1 mg/mL). Continue mixing for at least 24 h

at 4°C. Dilute to 0.4 mg/mL for the working solution.

2. Protein molecular-weight standards (Bio-Rad, Richmond, CA).

3. Acrylamide stock solution (30% acrylamide/0.8% bisacrylamide).

4. 4× Separating gel buffer: 0–1% 1.5 M Tris-HCl/0.1% SDS, pH 8.8. Dissolve 91 g of

Tris base and 2 g of SDS in 300 mL of H

O. Adjust to pH 8.8 with 1 N HCl. Add H

to 500 mL total volume.

5. 20% ammonium persulfate.

6. TEMED (N, N, N',N'-tetrametylethylenediamine).

7. Isobutyl alcohol.

8. 4× Stacking gel buffer: 0.5 M Tris-HCl/SDS, pH 6.8. Dissolve 6.05 g of Tris base and

0.4 g of SDS in 40 mL of H

O. Adjust to pH 6.8 with 1 N HCl. Add H

O to 100 mL total

volume.

9. 2× SDS/sample buffer: Dissolve 1.52 g of Tris base, 20 mL of glycerol, 2.0 g of SDS,

2.0 mL of 2-mercaptoethanol, and 1 mg of Bromphenol blue in 40 mL of H

O. Adjust

to pH 6.8 with 1 N HCl. Adjust to 100 mL with H

10. 5× SDS/electrophoresis buffer: Dissolve 15.1 g of Tris base, 72.0 g of glycine, and 5.0 g

of SDS in H

O, adjust to 1000 mL. Do not adjust the pH. It will be pH 8.3 when it becomes

diluted. Dilute 1/5 with H

O just before use (see Note 1).

2.2. HA-Substrate Gels

1. 3% Triton X-100 solution: 3% Triton X-100 in 50 mM HEPES, pH 7.4.

2. pH 3.7 hyaluronidase assay buffer: 0.15 M NaCl in 0.1 M formate buffer, pH 3.7.

3. pH 7.4 hyaluronidase assay buffer: 0.15 M NaCl in 50 mM HEPES, pH 7.4.

4. Alcian blue staining solution. Dissolve 0.5 g of Alcian blue in 100 mL of 3% acetic acid

solution.

5. Destaining solution for Alcian blue staining: 7% acetic acid.

Substrate Gel and Inverse Substrate Gel Technique 393

6. Coomassie blue solution: Dissolve 50 mg of Coomassie brilliant blue R (see Note 2) in

50 mL of methanol. Add 10 mL of acetic acid and 40 mL of H

O, mixing well.

7. Destaining solution for Coomassie blue staining: 50% methanol and 10% acetic acid.

2.3. Inverse HA-Substrate Gels

1. 3% Triton X-100 solution: 3% Triton X-100 in 50 mM HEPES, pH 7.4.

2. pH 7.4 hyaluronidase inhibitor assay buffer: Prepare 0.5 rTRU/mL bovine testicular

hyaluronidase (Sigma): 0.15 M NaCl, 1 mM MgCl

in 50 mM HEPES, pH 7.4 (see Note 3).

3. 0.1 mg pronase/mL in phosphate-buffered saline (PBS), pH 7.4.

4. Alcian blue solution. Dissolve 0.5 g of Alcian blue in 3% acetic acid solution.

5. Destaining solution for Alcian blue staining: 7% acetic acid.

6. Destaining solution for Coomassie blue staining: 50% methanol and 10% acetic acid.

7. PBS.

2.4. Equipment

1. Gel casting cassette.

2. Electrophoresis cassette.

3. 37°C incubator.

4. Rotating shaker platform.

5. Transilluminator.

6. Constant-voltage power supply.

7. Pipet.

8. Pasteur pipet or 10-mL syringes with size 22 gage needles.

9. Glass Petri dish, 15 cm diameter.

10. Razor blades.

3. Methods

3.1. Electrophoresis in HA Gel

1. Gel electrophoresis is performed using a modification of the method of Laemmli (16). Set

up the gel cassette.

2. Prepare the separating gel mixture by adding 5.0 mL of acrylamide stock solution, 3.75 mL

of HA solution (0.4 mg/mL), 3.75 mL of separating gel buffer, 2.5 mL of water, and 100 µL

of 20% ammonium persulfate.

3. Add 10 µL of TEMED. Gently swirl the flask to ensure even mixing. The addition of

TEMED will initiate the polymerization reaction, so it is advisable to work fairly quickly at

this stage.

4. Using a Pasteur pipet or syringe with a needle, transfer the separating gel mixture to the

gel cassette carefully until it reaches 1 cm below the bottom of the sample loading comb.

5. Add isobutyl alcohol slowly to the top of the gel (~1 cm) to smooth out the gel surface.

6. Allow the gel to polymerize for 20 min.

7. While the separating gel is setting, prepare the stacking gel solution. Mix 0.65 mL of

acrylamide stock solution, 1.25 mL of stacking gel buffer, 3.05 mL of water, and 50 µL of

20% ammonium persulfate.

8. When the separating gel has polymerized, pour off the overlaying isobutyl alcohol, and

then wash twice with H

O. Add 5 µL of TEMED to the stacking gel solution. Add the

stacking gel solution to the gel cassette until the solution reaches the cutaway edge of

the gel plate. Place the well-forming comb into this solution and leave to set. This will

394 Mio et al.

take about 15 min. It is useful at this stage to mark the position of the bottom of the wells

on the glass plates with a marking pen.

9. Carefully remove the comb from the stacking gel. Assemble the cassette in the

electrophoresis tank. Fill the top reservoir with electrophoresis buffer, ensuring that

the buffer fully fills the sample loading wells. Fill the bottom tank with electrophore-

sis buffer.

10. Mix samples with the same volume of 2× sample buffer and apply onto the gel (5–20 mL).

Protein molecular-weight standards should be also applied to the end lane of the gel.

11. Connect the electrophoresis cassette to the power supply using a current of 15 mA/gel.

Electrophoresis is stopped when the bromphenol blue reaches the bottom of the gel.

3.2. HA-Substrate Gel for the Detection of Hyaluronidase Activity

1. After electrophoresis, incubate the gel in 3% Triton X-100 for 1 h with agitation to remove

SDS from the gel.

2. Transfer gels into the hyaluronidase assay buffer using a suitable pH. Rinse the gel twice

with assay buffer.

3. Incubate on the rotating shaker for 16 h at 37°C.

4. Rinse the gel twice with distilled water.

5. Stain the gel in the Alcian blue solution for 1 h (see Note 4).

6. Place the gel in 7% acetic acid for destaining and fixation. Change solutions once every hour

until bands of hyaluronidase can be observed clearly. This step requires 2–3 h.

7. Rinse the gel twice with distilled water, followed by two washes of 50% methanol/10%

acetic acid.

8. Transfer the gel to the Coomassie blue solution. Incubate for 30 min.

9. Destain the gel with 50% methanol/10% acetic acid. Change solutions once every hour until

bands appear on the gel. This step requires 2–3 h.

10. Analyze the bands of hyaluronidase activity by comparing with the lane containing protein

molecular-weight standards. Hyaluronidase activity will appear as a white clearing on a pale

blue background. Protein bands will stain blue (see Note 6).

3.3. Inverse HA-Substrate Gel for the Detection of Hyaluronidase

Inhibitors

1. After electrophoresis, carefully separate the end lane containing the protein standards using

a razor blade. Protein standards are subjected to Coomassie blue staining. Perform the

following steps for the other portion of the gel.

2. Incubate the gel in 3% Triton X-100 in 50 mM HEPES, pH 7.4, with agitation to remove

SDS from the gel.

3. Rinse the gel twice with 50 mM HEPES, pH 7.4.

4. Gels are transferred to the appropriate hyaluronidase-containing solution. For the

detection of inhibitors of testicular hyaluronidase, e.g., 0.5 rTRU/mL of bovine testicular

hyaluronidase, pH 7.4 is used. Incubate in the reaction solution for 16 h at 37°C on the

rotating platform (see Note 5).

5. Rinse the gel twice with PBS.

6. Transfer the gel to a solution of 0.1 mg/mL pronase in PBS and incubate on the rotator for

4 h at 37°C. This step digests proteins that may produce false positives (see Note 6).

7. Rinse the gel twice with distilled water.

8. Transfer the gel to the Alcian blue solution, and stain the gel for 16 h.

9. Place the gel in 7% acetic acid for the destaining procedure. Change solutions once every

hour until hyaluronidase inhibitor bands can be observed. This step usually requires 2–3 h.