Purification and properties of hyaluronidase from bull sperm.

Hyaluronidase from bull sperm was fractionated by ammonium sulfate and further purified by DEAE-cellulose and Sephadex chromatography. The highly purified hyaluronidase preparation showed 2,370 units per mg of protein (68,730 N.F. units per mg of protein), i.e. 182-fold purification. Disc gel electrophoresis showed one major component. The molecular weight of bull sperm hyaluronidase was 62,000 by sodium dodecyl sulfate gel electrophoresis. Hyaluronidase from bull sperm has optimum activity at pH 3.8 and an absolute requirement for cations. Kplus and Naplus have a greater effect than Ca2plus, Mg2plus, and Mn2plus, whereas Co2plus, Cu2plus, and Zn2plus do not affect the enzyme activity. Purified preparations are less stable than crude extracts stored frozen at minus 15 degrees. Km of hyaluronidase with hyaluronic acid as substrate is 3.7 mg per ml and Vmax is 2.4 mumol per min by Hofstee plot.

Purified preparations are less stable than crude ettracts stored frozen at -15". K, of hyaluronidase with hyaluronic acid as substrate is 3.7 mg per ml and V,,, is 2.4 pmol per min by Hofstee plot.
Bovine testicular hyaluronidase has been purified to varying degrees of homogeneity and activity (l-4). Testicular hyaluronidase is localized esclusively in acrosomes of developing spermatids and spermatozoa as shown by immunofiuorescent staining of the intact cells (5) and by separation of sperm heads before disrupting acrosomes (6). Masaki and Hartree (7) observed that upon storage of normal bull semen hyaluronidase diffused from spermatozoa to the seminal plasma. They stated that the enzyme is localized in spermatozoa as vasectomy led to disappearance of hyaluronidase from the semen (8,9). Azospermic human semen is also devoid of the enzyme (10). Recently, Zaneveld et al. (11) reported a partial purification and properties of acrosomal hyaluronidase from frozen stored spermatozoa of the bull and showed that acrosomal hyaluronidase was identical with bull testicular hyaluronidase by immunodiffusion studies.
In view of the fact that most of hyaluronidase would have leaked into the seminal plasma on storage, \Tve compared the activity of the partially purified acrosomal hyaluronidase (11) to the activity of the enzyme present in the seminal plasma.
The activity of hyaluronidase in seminal plasma was extremely high.
For of Bull Sperm Hyaluronidase-Thawed bull semen was first centrifuged at 3,000 X 9 for 10 min to remove spermatozoa and the seminal plasma supernatant was subsequently centrifuged at 105,000 X g for 4 hours in order to remove the pellet containing the decapacitation factor (18). The supernatant from the high speed centrifugation was dialyzed for 48 hours at 4" against 0.01 M sodium phosphate buffer (pH 7.1) changing the buffer at 4,12, and 24 hours. The nondialyzable solution was brought to 30% saturation by adding solid (NH,)&04 at 4" and allowed to stand overnight.
After removal of the precipitate, the supernatant was brought to 65y0 saturation with (NH,)&O,, the mixture allowed to stand overnight at 4" and centrifuged at 10,000 X g for 30 min. The precipitate was dissolved in distilled water and dialyzed against distilled water. The insoluble precipitate that formed during the first 10 hours of dialysis was removed by centrifugation at 15,000 X g for 20 min and the resultant supernatant was lyophilized.
The lyophilized material (101 mg of protein) was dissolved in 0.02 M potassium phosphate buffer, pH 6.0, containing 0.15 M NaCl and applied to a column of Sephadex G-100 (2.5 X 93 cm) at 4" equilibrated with the same buffer (Column I).
The column was washed with 250 ml of the buffer at the rate of 24 ml per hour. The hyaluronidase-active fractions eluting in the first protein peak were pooled and concentrated to 20 ml. The concentrated solution (28 mg of protein) was applied to a DEAE-cellulose column (2.5 X 36 cm) at 4" equilibrated with 0.005 M potassium phosphate buffer, pH 7.1 (Column II).
The column was washed with 300 ml of the buffer and developed with a stepwise gradient of 0.05 M, 0.1 M, and 1 M NaCl at the rate of 27 ml per hour.
The hyaluronidase-active fractions eluting in the first protein peak with the buffer alone were pooled and concentrated by ultrafiltration (Amicon Diaflo, PM-10 filter) with several additions of 0.15 M NaCl solution at 4". The purified hyaluronidase obtained from above (3.3 mg) was applied to a Sephadex G-75 column (2.5 X 88 cm) which had been equilibrated with0.15 M NaCl at 4" (Column III).
The flow rate was adjusted to 16 ml per hour and 4-ml fractions were collected.
The hyaluronidase-active fractions appearing in the second protein peak were pooled and reapplied to a second column (2.5 X 36 cm) of Sephadex G-75 which had also been equilibrated with 0.15 M NaCl at 4" (Column IV). The flow rate was adjusted to 10 ml per hour and 2.5-ml fractions were collected.
The tubes containing hyaluronidase of constant specific activity were pooled (135 Mg). The scheme of purification is outlined in Fig. 1

Hyaluronidase
activity was negligible in the sedimented sperm (3,000 x g) and in the pellet of the second centrifugation (105,000 x g) compared to the seminal plasma.
From 159 ml of seminal plasma (76 mg per ml of protein), 143 ml (4.5 mg per ml of protein) of supernatant solution were obtained after centrifugation.
This protein was dissolved in distilled water and dialyzed against the same.
By removing the precipitate formed during dialysis, 66% of the contaminating proteins with little hyaluronidase activity were removed and 75% of the total hyaluronidase was recovered in the clear nondialyzable fraction. This step was essential for obtaining highly purified enzyme. The quantitative data on purification is summarized in Table  I. Using Column I the low molecular weight contaminants in the second peak were separated from hyaluronidase.
NaCl in the eluting buffer stabilized the enzyme.
Four components were separated by Column II.
The first peak eluted by buffer alone showed high hyaluronidase activity but the other three protein peaks had none. The fractions containing hyaluronidase (Column III) were pooled, concentrated by DiaAo, and applied to Column IV. The tubes having constant specific activity (Column IV, Fig. 2) were pooled, concentrated by Diaflo, and frozen.
If the Sephadex G-75 column (Column IV) is developed with distilled water according to the procedure of Borders and Raftery (4), the loss in activity is high as NaCl is essential to stabilize the purified enzyme.
NaCl is also helpful to dissociate hyaluronidase from contaminant resulting in good separation. Hyaluronidase obtained from Column IV (Fig. 2) was free from @-A-acetylglucosaminidase and P-glucuronidase activities. n .-*-~, hyaluronidase.
band was also observed on sodium dodecyl sulfate gel electrophoresis (Fig. 4).
Properties pH Optimum-Bull sperm hyaluronidase has an optimum at pH 3.8 aith a broad plateau of activity between pH 3.5 and pH 8.0. The enzyme is inactive at pH 2.6 and has less than 10% activity above pH 8.0. Ram sperm hyaluronidase showed a somewhat different pH activity curve (13). Cation E$ects--Hyaluronidase from bull sperm has a requirement for cations.
The effects of several mono-and divalent chlorides on enzyme activity are shown in Fig. 5. K+ and Na+ have a greater effect and at a wider range of concentration than Ca2+, Mg2+, and Mn*+, whereas, Coa+, Cu2+, and Zn2+ do not affect enzyme activity.
Zaneveld et al. (11) reported that Mg2+ and Ca2+ had no apparent effect on the activity of bull sperm acrosomal hyaluronidase at a fixed concentration and Fe2+ and Fea+ decreased the enzyme activity.
Using different concentrations of salts, our results showed that higher concentrations of ions inhibit enzyme activity. &c&Z&-Crude hyaluronidase at pH 7.0 is stable at room temperature for several days or frozen for several months. It is stable at 42" for 60 min, but loses 98% of the activity at 60" for 30 min.
Pure hyaluronidase (Column 1V) is not stable stored in solution at 4" for several weeks.
Kinetics-Kinetic studies performed at 37" with hyaluronic acid as substrate gave a K, of 3.7 mg per ml and V,,,,, of 2.4 pmol per min (Fig. 6).
Molecular Weight Determination-The mobility of the purified hyaluronidase (Column IV) compared to standards on sodium dodecyl sulfate gel electrophoresis (Fig. 4) gave a molecular weight of 62,000.

Hyaluronidase
is involved in the dispersal of the cells of the cumulus oiiphorus of the ovum by dissolving the intercellular matrix which is composed of hyaluronic acid aiding the entry of 3. Analytical gel electrophoresis of bull sperm hyaluronidase ourified bv the final step of gel filtration (Column IV) (A) was Compared "with commercially available enzyme (Worthington highly purified testicular hyaluronidase, HSEP) (B).
the spermatozoa through this barrier. Recently, it has been shown that species-specific antibodies against hyaluronidase can be developed that may be useful in preventing gamete interaction (21,22). In order to prepare species-specific antibodies pure hyaluronidase as obtained here would be very useful. We suggest that subscribers photocopy these corrections and insert the photocopies at the appropriate places where the article to be corrected originally appeared. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts.