Susan Stair Nawy Antonei B Csoka Kazuhiro Mio and Robert Stern

1. Introduction

Hyaluronidase is a term applied to a group of very dissimilar enzymes (1-3) that degrade hyaluronan (HA, hyaluronic acid), a high-molecular-weight glycosaminogly-can of the extracellular matrix. Some of these enzymes have the ability to degrade additional glycosaminoglycans, albeit at a slower rate. Most of the hyaluronidases from eukaryotes have both hydrolytic and transglycosidase activity, while those from bacteria operate by ^-elimination. HA is prominent whenever rapid cell proliferation and movement occur, particularly during embryogenesis, wound healing, repair and regeneration, and in tumorigenesis (4-8). Hyaluronidases regulate temporal and spatial distribution patterns of HA, critical during such processes. Hyaluronidases, often present at exceedingly low concentrations, are imbued with high but unstable specific activities. They can be difficult to detect, and their quantitation requires specialized techniques.

Previous methods for quantitation of hyaluronidase either lacked sensitivity, were slow and cumbersome, or required highly specialized reagents not available in most laboratories (9-12). This accounts in part for the relative neglect, until recently, of this important group of enzymes. An improved ELISA-like assay was developed in which hyaluronidase activity could easily be detected in most biological samples (13). The general technique is described here, together with notes regarding more specialized usage. The free carboxy groups of HA are biotinylated in a one-step reaction using biotin hydrazide. Standard and unknown samples of enzyme are subsequently allowed to react with the HA substrate covalently bound to the wells of 96-well microtiter plates. Residual substrate is detected with an avidin-peroxidase color reaction that can be read using a standard ELISA plate reader. A standard curve of hyaluronidase activity is run with each plate using serial dilutions of any hyaluronidase standard, and the 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 procedure 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 MgSO4 (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 dH2O and add to the HA-biotin solution to a final concentration of 30 ^M. Stir overnight at 4°C.

5. Remove the unlinked biotin and EDC by the addition of 4 M guanidine-HCl and dialysing against 1000x volumes of dH2O 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 dH2O with the bHA at a concentration of 0.2 mg/mL and pipet into 96-well Covalink-NH plates at 50 ^L per well.

Biotin Hydrazide
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).

2. Dilute EDC to 0.123 mg/mL in dH2O and pipet into the Covalink plates with the HA solution yielding a final concentration of 10 ^g/well HA and 6.15 ^g/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 MgSO4 (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 ^L/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 x 10-6 rTRU and assay 100 ^L/well, the standard enzyme reaction volume, in triplicate (see Note 4).

3. Pipet unknown samples into plate, 100 ^L/well, in triplicate. Preliminary experiments must be performed to determine the proper dilution of the enzyme preparation. In order to establish 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.2. A four-parameter curve-fit of bovine testicular hyaluronidase assays as standard reactions performed at pH 3.7, diluted from 1.0 to 1 x 10-6rTRU/well. (Reprinted by courtesey of Academic Press from ref. 14.)

6. Terminate the reaction by addition of 100 ^L/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 ^L/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 ^L/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 ^L of 30% H2O2.

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 detergents, 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 undertaken 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 concentrations 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. Occasionally, extracts must be diluted out. The inhibitors appear to occur at more limiting concentrations than enzyme. Paradoxically, greater levels of apparent hyaluronidase activity are observed in plasma samples that are more diluted.

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.

Time (min)

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.

4. Some hyaluronidase inhibitors are Mg2+-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 standardizes 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.

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 laboratory procedure and, requiring only 1-^L 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 generated by each cleavage reaction. On the other hand, leech hyaluronidase, which is an endo-p-glucuronidase rather than an endo-p-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 p-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 p-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.

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