THE JOURNAL OF BIOLOGICAL CHEXISTRY Vol. 243, No. 13, Issue of July 10, pp. 3756-3762, 1968

Printed in U.S.A.

Purification and Partial Characterization of

Testicular Hyaluronidasc”

(Received for publication, February 12, 1968)

CHARLES L. BORDERS, JR.,$ AND M. A. RAFTERY

From the Department of Chemistry, California Institute of Technology, Pasadena, California 91109

SUMMARY

A new method is presented for the purification of testicular hyaluronidase involving ion exchange chromatography fol- lowed by gel filtration on Sephadex G-75. A highly purified hyaluronidase preparation has been obtained which contains 45,000 National Formulary activity units per mg, dry weight, and which migrates as a single component on polyacrylamide gel electrophoresis at pH 4.3. The enzyme has been shown to be a glycoprotein containing 5.00% mannose and 2.17% glucosamine (expressed as IV-acetylglucosamine). The molecular weight of the purified hyaluronidase has been determined to be 61,000 by gel filtration methods. This value is in contrast to a molecular weight of 126,000 for the crude enzyme, as determined by similar techniques. This difference between the crude and purified enzyme could be due to association with a carrier protein in the crude tes- ticular extract or to the existence of hyaluronidase as a dimer in the crude state. Only 76% of the weight of the purified hyaluronidase could be accounted for by its amino acid and carbohydrate composition.

There have been several studies in the past few years on the purification and characterization of testicular hyaluronidase (l-4). Brunish and Hogberg (1) prepared a highly active enzyme containing approximately 40,000 i.u. per mg. They found their sample to be a glycoprotein, but did not identify the types of neutral sugar and amino sugar residues involved. They reported a molecular weight of 43,200 as determined by the Archibald approach to sedimentation equilibrium method, but found their sample to be somewhat heterogeneous in the ultracentrifuge. They reported an amino acid analysis based on a single determination, and were unable to show the homo- geneity of their preparation. Soru and Ionescu-Stoian purified the enzyme with a Sephadex-DEAE-Sephadex chromatography

* This investigation was supported by United States Public Health Service Grant GM-14452-01. Contribution 3647 from the California Institute of Technology.

lSst7F” t’ a lonal Institutes of Health Predoctoral Fellow, 1966 to

system, and obtained hyaluronidase of very low activity (2, 3). They reported an amino acid composition for their preparation which was radically different from that of Brunish and Hogberg, and were unable to detect any carbohydrates (4). The homo- geneity of their enzyme preparation was investigated only by paper electrophoretic methods (3), and the material had a low specific activity.

Because of the various findings from different laboratories on the chemical and physical properties of testicular hyaluronidase, the present investigation was undertaken in an attempt to isolate and characterize the enzyme more thoroughly,

METHODS

MaterialsCrude hyaluronidase (type I, Lot 27B-1200, 280 National Formulary units per mg), hyaluronic acid (type III, Lot 36B-2460-l), bovine serum albumin (Fraction V), ribonu- clease (bovine pancreas, five times crystallized, type I-A), blue dextran (B-2000), and Sephadex G-75 and G-100, both in bead form (10 to 40 p), were obtained from Sigma. Highly purified hyaluronidase (Lot HSEP 6LB, 3450 U. S. P. units per mg) and chymotrypsinogen (crystallized, salt-free) were obtained from Worthington. Bio-Rex 70 (minus 400 mesh) was obtained from Calbiochem. Egg albumin (crystallized) was obtained from Armour. National Formulary Standard hyaluronidase was obtained from the National Formulary Reference Standards section of the American Pharmaceutical Association. All other chemicals, reagent grade, were obtained from commercial sources.

To minimize inactivation of the purified hyaluronidase by contact with glass (5), polyethylene test tubes were used in the preparative work and in all the assays of enzymatic activity.

/4ssuy-Hyaluronidase activity was determined turbidimet- rically by a modification of the method described in the Worth- ington 2MunuuZ. The substrate solution was made up to con- tain 0.40 mg of hyaluronic acid per ml in 0.1 M NaH2P04, 0.15 M NaCl (pH 5.3). To 0.50 ml of the substrate solution was added 0.45 ml of the pH 5.3 buffer and the mixture was preincu- bated at 37” for at least 0.5 hour. Then an appropriate amount of enzyme (0.05 to 2.0 pg) in 0.05 ml of buffer was added and the mixture was incubated for 10.0 min at 37”. The reaction was stopped by dilution with 9.0 ml of albumin reagent buffered at pH 4.2 with 0.5 M acetate. After 10 min the optical density

3756

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at 420 rnp was read and the activity was expressed in National the data obtained after hydrolysis for 20 and 70 hours in 6 N

Formulary units by comparison with a st,andard curve made up HCl in a linear fashion and ext,rapolating to zero hydrolysis with known am0unt.s of National Formulary standard hyaluron- time. Data obtained from the 70-hour hydrolyses were used to idase. obtain values for valine, leucine, and isoleucine. The tyrosine-

Purification of Hyaluronidase-Protein was determined by tryptophan ratio was determined in 0.1 N NaOH by the method the method of Lowry et al. (6), with a standard hyaluronidase of Bencze and Schmid (9). Cystine plus cysteine was also which had been purified by Bio-Rex 70 chromatography, as determined as carboxymethylcysteine after reduction and described below. This first step in the purification procedure carboxymethylation according to the method of Crestfield, is a modification of the method of Rasmussen (7). Crude hy- Moore, and Stein (10). aluronidase (Sigma, type 1, 25.88 g) was dissolved in 750 ml of Neutral sugars were quantitatively determined by the orcinol 0.1 M NaH2P04, pH 6.0. The pH was adjusted to 5.9 with 50% test described by Hartley and Jevons (II), with mannose as a NaOH and the solution was applied to a column of Bio-Rex 70 standard. Neutral sugars were qualitatively identified by (minus 400 mesh), H+ form, 6.0 x 43 cm, pre-equilibrated with descending chromatography on Whatman No. 1 paper for 24 0.1 M NaH2P04, pH 6.0. The column was set up in a constant hours with ethyl acetate-pyridine-water (12:5:4) as solvent temperature enclosure at 14”. After all of the protein solution (12). They were identified by comparison with simultaneously had been applied, the column was washed with 4 liters of 0.1 run standards. The sugars were detected by the AgN03- M NaHzP04, pH 6.0, at a rate of 150 to 200 ml per hour. The NaOH method (13) with destaining and preserving by treat- eluting buffer was changed to 0.3 M Pod, pH 7.7, and 12-ml ment with NazSz03. For analysis of carbohydrates, 1 mg of fractions were collected. The protein concentration of each the purified enzyme was dissolved in 1 ml of 1 N HCI and hy- fraction and the hyaluronidase activity of various fractions were drolyzed at 108” for 3 hours in an evacuated sealed tube. After determined. The tubes containing hyaluronidase activity were drying, the hydrolysate was taken up in 1 ml of water and ap- pooled, dialyzed against six changes of distilled water at 4” for plied to a column (0.9 X 10 cm) of Dowex 50-X8, 200 to 400 a total of 30 hours, centrifuged to remove a small amount of mesh, H+ form, and eluted with water, and the first 30 ml were insoluble material, and lyophilized. evaporated to dryness on a rotary evaporator with a bath

The purified hyaluronidase obtained above (2.10 g) was dis- temperature of 35”. The dried neutral sugars were taken up solved in 50 ml of 0.1 M NaCl and applied to a Sephadex G-75 in 2.0 ml of water and a l.O-ml aliquot was used for quantitative column (5.0 x 76 cm) which had been pre-equilibrated with analysis while the remainder was used for qualitative tests. 0.1 M NaCl at 4”. The flow rate was adjusted to 50 ml per hour Molecular Weight Determination-Molecular weight estima- and lo-ml fractions were collected. Protein and hyaluronidase tions were made on Sephadex G-100 (bead form) by the method activity were located and the appropriate tubes were pooled of Andrews (14). A column (1.5 x 91.5 cm) of Sephadex G-100 and reapplied to a second column (5.0 x 72 cm) of Sephadex was equilibrated at 20” with 0.10 M NaHzPO+ pH 5.3, con- G-75 which had also been pre-equilibrated with 0.1 M NaCl at taining 0.15 M NaCl and saturated with chloroform. This 4”. Protein and hyaluronidase activity were again located and buffer was chosen to approximate the conditions of the assay the appropriate tubes were pooled and applied to a column used to determine hyaluronidase activity. The column was (5.0 x 70 cm) of Sephadex G-75 which had been pre-equilibrated standardized with blue dextran, bovine serum albumin, bovine with distilled water at 4”. The flow rate was adjusted to 30 ml serum albumin dimer, ovalbumin, chymotrypsinogen, and ribo- per hour and lo-ml fractions were collected. The protein and nuclease A. Purified hyaluronidase was run simultaneously hyaluronidase activity were located as described above, and it with the standard proteins. Fractions of 1.0 ml were collected; was determined that the major protein peak came out just the standard proteins were located by reading optical densities ahead of the NaCl by locating the latter with AgN03. The at 280 rnp, while the hyaluronidase was located by activity tubes of constant hyaluronidase activity were pooled and lyoph- measuremen&. Duplicate runs of different hyaluronidase ilized to give a highly purified preparation of hyaluronidase. concentrations were made. Crude hyaluronidase was run with

Disc Electrophoresis-Disc electrophoresis at pH 4.3 was the blue dextran marker, and was located by activity measure- carried out by the method of Reisfield, Lewis, and Williams (8), ments. with a 15oj, polyacrylamide gel, omitting all but the small pore gel, and applying the samples in 10% sucrose solution. Elec- RESULTS

trophoresis was carried out at 4 ma per tube for 90 min. The gels were stained with 1% Amido schwarz in 7 To acetic acid and Purification

destained by washing with 7% acetic acid. Bio-Rex 70 Chromatography-When the crude hyaluronidase Amino Acid and Carbohydrate Analytis-Acid hydrolyis of was applied to Bio-Rex 70 equilibrated with 0.1 M NaH2PO+

hyaluronidase prior to amino acid analysis was carried out with pH 6.0, most of the protein passed directly through the column 6 N HCl for 20 and 70 hours at 108” in evacuated sealed tubes. with a large amount of gold-colored impurity. When the eluting The amino acids were determined on an automatic amino acid buffer was changed to 0.3 M NaHzPOh, pH 7.7, about 5 liters analyzer (Beckman-Spinco model 120B) on a column, 0.9 x 50 were passed through before the hyaluronidase was eluted as a cm. Amino sugars were determined on the same column after broad peak (Fig. 1). The fractions indicated by the arrow were hydrolysis for 20 hours with 1 N HCI at 108” in evacuated sealed pooled and after dialysis and lyophilization yielded 2.13 g of tubes. In the chromatographic system used they were eluted hyaluronidase with an activity of 2300 National Formulary after phenylalanine. As a check, amino sugars were also deter- units per mg, dry weight. mined by linear extrapolation of the results obtained after First Sephadex G-76 Chromatography in 0.1 M NaCl-When hydrolysis in 6 N HCl for 20 and 70 hours to zero hydrolysis the protein material obtained from the Bio-Rex 70 chromatog- time. Threonine and serine were also determined by plotting raphy was run on a Sephadex G-75 column equilibrated with

Issue of July 10, 1968 C. L. Borders, Jr., and M. A. Raftery 3757

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r I I I I 1

dase had shown that it was indeed retained on Sephadex in the absence of salts, it was thought that a step such as this might

2 6.0

-2 -PROTEIN be a valuable one in the purification scheme. That this was

c -ENZYME ACTIVITY

A +‘ooo indeed the case is shown in Fig. 3. A small amount of material devoid of hyaluronidase activity was eluted in the position at which hyaluronidase normally was eluted in the presence of 0.1 M

NaCl. This was followed at a distance by a much larger peak, the front part of which was devoid of hyaluronidase activity and the back part of which was highly purified hyaluronidase. The fractions indicated by arrows had an essentially constant ac- tivity of 45,500 National Formulary units per mg. When it was determined that the hyaluronidase had been eluted just ahead of the NaCl peak, the fractions of constant activity were pooled and lyophilized in a polyethylene container. The ly- ophilized material was stored in a desiccator below 0”. It had

EFFLUENT .VOLUME, ml. an activity of 45,000 National Formulary units per mg, dry

FIG. 1. Chromatography of crude hyaluronidase on Bio-Rex weight, and retained its activity even after storage as a frozen 70. The hyaluronidase was applied in 0.1 M NaHtPOd, pH 6.0, solution in distilled water for 4 months. ,4 summary of the and eluted with 0.3 M NaHzPOd, pH 7.7. The details of the chro- matography are described in the experimental section of the text.

various steps in the purification is given in Table I.

Disc Electrophoreti I 1 I I I I I I I

I 00,000 The homogeneity of the purified hyaluronidase is indicated by the presence of a single band in polyacrylamide gel elec-

-PROTEIN trophoresis at pH 4.3 (Fig. 4). Hyaluronidase at various - ENZYME ACTIVITY stages of purification is shown and the degree of purification is

f 80,000 , evident. It is interesting to compare the hyaluronidase pre-

r pared as described herein with a preparation obtained by the

i method of Soru and Ionescu-Stoian (2, 3) (purchased as highly 3 L purified hyaluronidase from Worthington) (Fig. 5) which was i

k 1 , , ~ ,,=~ k s

40,000 shown to contain four major components and at least six minor

A.&- components by disc electrophoresis.

Amino Acid and Carbohydrate Composition

-( 1 The amino acid and carbohydrate composition of hyaluroni- 0 200 4c )O 600 800 dase is given in Table II. The values are expressed as millimoles

EFFLUENT VOLUME, ml of residue per 100 g of enzyme since only 76% by weight of the FIG. 2. Chromatography of partially purified hyaluronidase enzyme could be accounted for as amino acids and carbohydrates.

on Sephadex G-75 in 0.1 M NaCl solution. The details of the chromatography are described in the experimental section of the

This is analogous to the preparation of Brunish and Hiigberg

text. (1) in which only 75% by weight of their preparation could be accounted for as amino acids and carbohydrates. As a check

3758 Purification of Testicular Hyaluronidase Vol. 243, No. 13

0.1 M NaCl, the major part of the hyaluronidase activity was against contamination by salt from the final Sephadex G-75

eluted in a trough between two major inactive components chromatography, a small amount of purified hyaluronidase was

(Fig. 2). The fractions indicated in Fig. 2 were pooled and dialyzed extensively against distilled water at 4” at a concentra-

yielded hyaluronidase with an activity of 21,600 National Formulary units per mg. These pooled fractions were used in the next step. That the Bio-Rex 70 chromatography was essential in the purification procedure was indicated by the fact

2 0.60 I- i

30,000

that when crude commercial hyaluronidase was applied directly to a Sephadex G-75 column equilibrated with 0.1 M NaCl, only a 1.5-fold purification resulted.

Second Sephaclex G-75 Chromatography in 0.1 M NaCl-When the chromatography on Sephadex G-75 equilibrated with 0.1 M NaCl was repeated, additional purification resulted. The fractions of highest hyaluronidase activity were pooled and yielded hyaluronidase with an activity of 31,000 National Formulary units per mg. These pooled fractions were used directly in the next step. EFFLUENT VOLUME, ml.

Sephadex G-75 Chromatography in Water-It is well known that many basic proteins are held up by Sephadex gels at very

FIG. 3. Gel filtration of partially purified hyaluronidase on

low ionic strength. Since previous experience with hyaluroni- Sephadex G-75 in water. The details of the chromatography are given in the experimental section of the text.

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Issue of July 10, 1968 G. L. Borders, Jr., and, M. A. Rajtery

TABLE I

3759

Summary of p?tri$cation of testicular hvaluronidase

Crude enzyme ............................ Bio-Rex 70 .......................... : .... First Sephadex G-75 in 0.1 M NaCl. ...... Second Sephadex G-75 in 0.1 M NaCl ..... Third Sephadex G-75 in water ............

- I

ml

40 70 90

Amount recovered Relative activity Yield

mg protein/m1

2.85 0.89 0.32

mg Protein '.F.a units X IO ‘3. wds/mg pro1ein % 25880 7.25 280 1.0 100 2130 4.90 2300 8.2 67.6

114 2.48 21600 77 34.2 62 1.93 31100 111 26.6 28.8 1.31 45500 163 18.1

0 National Formulary.

tion of 1 mg per ml, without precipitation or loss of activity. Amino acid and sugar analyses of this dialyzed enzyme again showed that only about 75% of its weight could be accounted for as amino acids and carbohydrates. It should be pointed out that the data were not corrected for moisture or ash (salts) in the sample, or for destruction of amino acid residues during hydrolysis under the influence of carbohydrates present in hydrolysis mixtures.

In comparing our analyses with that of Brunish and Hogberg,

FIG. 4. Disc electrophoresis patterns of hyaluronidase prepara- tions at various stages of purification. A, 50 pg of highly puri- fied hyaluronidase with an activity of 45,000 National Formulary units per mg. B, 300 rg of hyaluronidase which had been purified by Bio-Rex 70 chromatography and which had an activity of 2,300 National Formulary units per mg. C, 600 pg of crude com- mercial hyaluronidase with an activity of 280 National Formulary units per mg. The details of the electrophoresis are given in the experimental section of the text.

FIG. 5. Disc electrophoresis patterns obtained from (A) 50 rg of highly purified hyaluronidase with an activity of 45,000 National Formulary units per mg and (B) 300 rg of highly purified hyaluronidase prepared by the method of Soru and Ionescu-Stoian (2, 3) with an activity of 3,450 National Formulary units per mg. The details of the electrophoresis are given in the experimental section of the text.

the largest discrepancies lie in the data for cystine and glucosa- mine. These authors found 3.12% half-cystine, while we find lSQa/,. To check our value, cystine plus cysteine were deter- mined as carboxymethylcysteine after reduction and carboxy- methylation by the method of Crestfield et al. (10). This gave a value for half-cystine which was within 2’% of the previously determined value. It may be pointed out that the amino acid data of Brunish and Hogberg are based on a single deter- mination, which may account for the observed difference. For the amino sugar content of the enzyme, Brunish and Hiig- berg reported a value of 5.0% by wet chemical determina- tion, while we found glucosamine to be present in an amount

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3760 Vol. 243, No. 13 PuriJication of Testicular Hyaluronidase

TABLE II Amino acid and carbohydrate composition of purified hyaluronidase

The 20- and 70-hour data are the average of three and two runs, respectively. Valine, isoleucine, leucine, serine, threonine, tryp- tonhan. ducosamine. and mannose were determined as described in the text. The ammonia data was not corrected for contribution because of the destruction of glucosamine.

Residue Hydrolysis data

20.hr I 70.hr

Lys .......... His. ......... Arg. ......... Asp ......... Thr .......... Ser. ......... Glu .......... Pro .......... Gly .......... Ala. ......... c yy ..........

Val Met ...................... Ile ......................... Leu ........................ Tyr ........................ Phe. ....................... NH3. ..................... Try ........................ Glucosamine (as N-acetyl-

glucosamine) ............ Mannose. .................. Hexosamine-HCl. .......... Neutral sugars .............

Total. 75.8Og

25.9 zt 1.9 9.3 f 0.8

19.5 f 1.6 47.1 f 2.5 22.6 f 0.6 33.1 f 1.3 34.8 f 0.8 25.7 f 1.9 25.4 f 1.1 24.3 f 0.6 12.7 f 2.3

29.2 f 0.3 5.7 f 1.0

15.1 f 0.7 37.7 f 0.4 17.1 f 0.4 17.1 f 1.5 65.8 f 4.0

25.7 f 0.4 8.8

19.3 f 0.7 47.3 f 0.6 22.0 zt 0.8 28.7 f 0.3 35.9 f 0.3 25.4 f 0.4 26.6 f 0.3 24.3 f 0.7 13.0 f 1.4

30.6 f 0.2 5.1 f 0.5

17.6 f 0.3 37.5 f 0.4 16.6 f 0.3 16.9 f 1.0

Average or extrapolated

Value Composition of hyaluronidase

25.8 37.2 4.77 9.2 13.3 1.82

19.4 28.0 4.37 47.2 68.0 7.83 22.8 32.9 3.34 34.9 50.3 4.38 35.2 50.7 6.55 25.6 36.9 3.58 25.9 37.3 2.13 24.3 35.0 2.49

12.9 18.6 1.89

30.6 5.5

17.6 37.6 16.9 17.0 65.8

(13.0)

44.1 4.37 4.20 7.9 1.04 0.64

25.4 2.87 2.73 54.2 6.13 5.66 24.4 3.98 3.22 24.5 3.61 3.29

(7.4) (21.4)

(18.7)

(10.7)

3.48

(30.8) 2.17 5.0

-

Brunish and H6igberg (1)

4.21 1.51 3.39 7.78 2.84 3.74 6.36 3.27 2.09 2.55 3.12

2.43

5.0 5.2

of 2.17% (assuming it to be N-acetylglucosamine in the intact enzyme). Our value was obtained by two different methods: (a) by determination after hydrolysis in 1 N HCl and (b) by extrapolation of the 6 N HCl hydrolysis data (20- and $0.hour hydrolysis times) to zero hydrolysis time. These methods gave the same value to within ~5%. The absence of galactosamine and mannosamine was confirmed by the chromatographic analyses.

Brunish and HGgberg reported a 5.2% neutral sugar content in their preparation. As shown in Fig. 6, we were able to detect only mannose, and in the amount of 5.0%. The paper chromatogram depicted in Fig. 6 indicates the absence of uranic acids, pentoses, and methylpentoses. For this reason, as well as the small amount of material available to us, no further tests for these sugars were performed.

Molecular Weight

When 0.012 mg of purified hyaluronidase was chromatographed with 3 mg each of bovine serum albumin, ovalbumin, and ribonu- clease A, 1.5 mg of chymotrypsinogen, and a trace of blue dextran on a Sephadex G-100 column (1.5 x 91.5 cm), the hyaluronidase was eluted immediately following serum albumin. When V, against log (molecular weight) was plotted for the standard proteins in the mixture (Fig. 7), the molecular weight of the purified hyaluronidase was estimated as 61,000. In a separate

determination with 0.24 mg of purified hyaluronidase with the standard proteins described above, the molecular weight was again determined to be 61,000.

In contrast to the elution pattern of the purified hyaluroni- dase, when crude commercial hyaluronidase was run on the same column and located by activity measurements, its molecular weight was determined to be 126,000. It is most likely that the Bio-Rex 70 chromatography step in the purification procedure was responsible for the lower molecular weight observed for the purified enzyme, since hyaluronidase which had been partially purified by Bio-Rex 70 chromatography and the first Sephadex G-75 chromatography was shown to yield a peak of enzymatic activity on the Sephadex G-100 column which corresponded to a molecular weight of 61,000.

DISCUSSION

One of the main advantages of the purification procedure described in the present instance is that the handling of the purified hyaluronidase at various stages of purification was kept at a minimum. It is known that dialysis of highly purified hyaluronidase at low concentrations of enzyme causes consid- erable denaturation (1). In the procedure described herein, dialysis of highly purified hyaluronidase was avoided, and thus losses by denaturation were minimized.

Although the specific activity, amino acid composition, and

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Issue of July 10, 1968 C. L. Borders, Jr., and ill. A. Rajtery 3761

carbohydrate composition of the purified hyaluronidase obtained as described in this paper are not inconsistent with those de- scribed by Brunish and Hogberg (l), they are in disagreement with those reported for the preparation of Soru and Ionescu- Stoian (24). Since the preparation described here has a much higher specific activity than that of the material of Soru and Ionescu-Stoian, and since disc electrophoresis of this preparation indicates a highly purified material while such electrophoresis of a sample prepared by the method of Soru and Ionescu-Stoian shows heterogeneity, it is obvious that the preparation described in this communication is more representative of the native enzyme in highly purified form.

Attempts were made to use the method of Hogberg (15) as the preliminary purification step of our procedure. However, hyaluronidase with a specific activity of only 3,000 units per mg was obtained. Brunish and Hogberg reported a prepara- tion obtained by this method to have a specific activity of 20,000 units per mg. The reason for the discrepancy is not known. Since only 40% of the hyaluronidase activity was recovered by the method of Hogberg (15), in our hands, while 65% re-

FIG. 6. Qualitative determination of the neutral sugars present in purified hyaluronidase by paper chromatography. A, standard mixture of sugars: 1, mannose; 8, glucose; 8, galactose; 4, man- nosamine; 6, glucosamine. B, neutral sugars present in purified hyaluronidase; only mannose is observable. Details of the nro- cedure are given in the experimental section of the text. *

300 I I I I I

200-o BLUE DEXTRAN

IOO- d 80-

‘0 60-

x !E 40-

(3 30- w 3 20-

% 3 IO- Y 8-

6-

4- HYALURONIDASE -

3

2 CHYMOTRYPSINOh, ;

RIBONUCLEASE A\ I I I I I I

40 60 80 100 Ve , ml.

FIG. 7. Molecular weight determination of crude hyaluronidase and of highly purified hyaluronidase by gel-filtration on Sephadex G-100 columns. Details of the chromatographic procedure and treatment of data are given in the experimental section of the text.

covery was attained with the method of Rasmussen (7), the latter was used as the preliminary purification step. The material obtained by the method of Hogberg (15) was not tested by Sephadex G-75 chromatography.

There has been disagreement in the literature regarding the molecular weight of hyaluronidase. In studying a sample prepared by the method of Hogberg (15), Malmgren (16) re- ported a molecular weight of 11,000, based upon sedimentation velocity and diffusion data and a value of 14,000 with the Archibald principle of approach to sediment.ation equilibrium. Brunish and Hiigberg, on the other hand, reported a molecular weight of 43,200 for their preparation as determined by the Archibald method and extrapolation to zero centrifugation time (1). They noted, however, that the apparent molecular weight decreased with increased time of centrifugation, and attributed this phenomenon to a heterogeneous hyaluronidase sample.

When the molecular weight of the purified hyaluronidase described in this communication was determined by the method of Andrews, a value of 61,000 was obtained. This value was independent of concentration over a 20-fold range, indicating that the concentration dependence of the molecular weight value is negligible. This value of the molecular weight of the purified enzyme is in sharp contrast to the value of 126,000 obtained for the crude hyaluronidase preparation as determined by the same method. One plausible explanation of this differ- ence in the crude and purified hyaluronidases is that in the crude extract the enzyme is associated with a carrier protein as postu- lated by Malmgren (16). Another explanation of this difference is that the enzyme exists as a dimer in the crude extract and a monomer in the purified form, and that the Bio-Rex 70 chroma- tography step removes a cofactor, possibly a divalent cation, which is necessary for the dimerization.

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3762 PuriJication of Testicular Hyaluronidase Vol. 243, No. 13

An unresolved aspect of our investigation is that only 76y0 of 2. SORU, E., AND IONESCU-STOIAN, F., Biochim. Biophys. Acta,

the weight of the protein could be accounted for by constituent 69, 538 (1963).

amino acids and carbohydrates. This result does not seem 3. SORU, E., AND IONESCU, STOIAN, F., J. Chromatogr., 17, 538

artifactual since a similar result was obtained on analysis after prolonged dialysis of the purified hyaluronidase against dis- tilled water. Also, Brunish and Hiigberg were able to account for only 75% of the weight of their preparation as amino acids and carbohydrtites, although their data were based on a single determination (I). The values obtained in our investigation were not corrected for moisture, but it seems unlikely that our preparation contains up to 25 y0 moisture.

4. (1965).

In conclusion, the results presented show that testicular hyaluronidase can be isolated from crude extracts as a highly purified preparation by use of a simple ion exchange step, followed by gel filtration. The resulting preparation has been shown to possess a higher specific activity with hyaluronic acid as substrate than previously reported preparations. It has been further shown to possess a molecular weight of 61,000, in contrast to previous conflicting reports. Whether the purified enzyme is composed of subunits is at present being studied.

REFERENCES 1. BRUNISH, R., AND H~GBERG, B., Compt. Rend. Trav. Lab.

Curlsberg, 32, 35 (1960).

5. 6.

7. 8.

9. 10.

11.

12.

13.

14. ANDREWS, P., Biochem. J., 91, 222 (1964). 15. H~GBERG, B., Acta Chem. &and., 6, 1098 (1954).

16. MALMGREN, H., Biochim. Biophys. Acta, 11, 524 (1954).

SORU, E., AND IONESCU-STOIAN, F., Arch. Roumaines Path. Exp. Microbial., 22, 783 (1963); Chem. Abstr., 60, 9547e (1964).

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Charles L. Borders, Jr. and M. A. RafteryPurification and Partial Characterization of Testicular Hyaluronidase

1968, 243:3756-3762.J. Biol. Chem. 

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