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 MgCl2 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.
1. Gel casting cassette.
2. Electrophoresis cassette.
4. Rotating shaker platform.
6. Constant-voltage power supply.
8. Pasteur pipet or 10-mL syringes with size 22 gage needles.
9. Glass Petri dish, 15 cm diameter.
10. Razor blades.
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 ^L of 20% ammonium persulfate.
3. Add 10 ^L 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.
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 ^L of 20% ammonium persulfate.
8. When the separating gel has polymerized, pour off the overlaying isobutyl alcohol, and then wash twice with H2O. Add 5 ^L 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 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 2x 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 mMHEPES, 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).
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.
Fig. 1 Detection of hyaluronidase and hyaluronidase inhibitors using HA-substrate gel and inverse HA-substrate gel procedures. M), molecular-weight markers. Lanes 1 and 2: HA-substrate gel for the detection of hyaluronidase activities. Examination of bovine testicular hyaluronidase, PH-20 (lane 1), and human plasma hyaluronidase, Hyal-1 (lane 2), were performed at pH 7.4 and pH 3.7, respectively. Lanes 3 and 4: Inverse HA-substrate gel for the examination of hyaluronidase inhibitor in mouse serum. The HA-containing gel, to which mouse serum had been applied, was digested with 0.5 rTRU/mL of bovine testicular hyaluronidase at pH 7.4 (lane 3). The position of the hyaluronidase inhibitors corresponds to a band in which the HA remained undigested (lane 3, A). A false positive corresponds to a band of endogenous plasma glycoprotein and possible HA-binding protein. This band could be identified by running a corresponding gel that does not contain HA (lane 4, B). Lane 1, 0.5 rTRU bovine testicular hyaluronidase; lane 2, 0.5 ^L human plasma; lanes 3 and 4; 4 ^L human plasma.
10. Rinse the gel twice with distilled water.
11. Establish the molecular sizes of hyaluronidase inhibitors by comparison with protein molecular-weight standards.
1. This protocol is designed for two Bio-Rad minigels (8 x 10 cm.). For other sizes or thicknesses, volumes of stacking and separating gels, and operating current, must be adjusted accordingly.
2. Not all Coomassie blue preparations work equally well. We have found the product of BDH Chemicals, (Poole, UK) optimal. There are others that do not work at all.
3. Analysis of bovine testicular hyaluronidase on an HA-substrate gel demonstrates two forms of the enzyme (see Fig.1, lane 1) corresponding to the soluble and membrane-bound forms of the enzyme (8,9). Analysis of human serum on the HA-substrate gel performed at pH 3.7 reveals a band at 57 kDa, which is Hyal-1 (see Fig. 1, lane 2). On an inverse HA-substrate gel performed at pH 7.4, two bands, at 120 and 83 kDa, are identified in mouse serum (see Fig. 1, lane 3). The data can be compared using the pattern obtained from a conventional HA-free gel (see Fig. 1, lane 4). The 83-kDa band persists, demonstrating that this is an artifact, corresponding to an endogenous plasma glycoprotein. The 120-kDa band corresponds to one of the hyaluronidase inhibitors in mouse serum, an inhibitor of neutral-active PH-20 enzyme.
4. Alcian blue is commonly used for staining HA (17-21). Acidification of the Alcian blue is recommended. This enhances staining and prevents precipitation of dye. For optimal kDii M
results, staining should be performed for 16 h in a solution of 0.5% Alcian blue in 3% acetic acid. When sequential staining with Alcian blue and Coomassie blue is performed, staining for 1 h with Alcian blue is sufficient. For the inverse substrate gel procedure, a single staining step with Alcian blue is recommended.
5. The hyaluronidase digestion step in the inverse HA-substrate gel procedure is obviously critical but can be difficult, as the gel is very fragile at this stage. Enzyme activity can also vary with minor changes of pH and temperature. Optimization of the concentration of hyaluronidase in the gel digestion step may be required with each experiment. We routinely utilize three different levels of hyaluronidase with each experiment (0.25, 0.50, and 2.00 rTRU/mL) to avoid over- and underdigestion.
6. High levels of a protein can prevent dye penetration into the gels (9,10) and can generate false positive bands. Albumin introduces such an artifact when plasma and serum samples are examined. Albumin is also a HA-binding protein (22-24), which may explain its persistence in these HA-containing gels. Pronase treatment of the gels eliminates such false positives. Proteins present in lesser amounts will appear as blue bands following Coomassie blue staining. These should disappear if a pronase digestion step is interposed.
This work was supported by Lion Corporation, Kanagawa, Japan, to K. M., and by National Institutes of Health (USA) Grant 1P50 DE/CA11912, to R. S.
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