The purification and some properties of pig liver hyaluronidase

Biochimica et Biophysica Acta 838 (1985) 257-263 Elsevier

257

BBA21972

The purification and some properties of pig liver hyaluronidase Marina B. Joy, Kenneth S. Dodgson, Anthony H. Olavesen and Peter Gacesa * Department of Biochemistry, University College, P.O. Box 78, Cardiff, CF1 1XL (U.K.) (Received July 20th, 1984)

Key words: Hyaluronidase; Glycosaminoglycan; (Pig liver)

Hyaluronidase (hyaluronate 4-glycanohydrolase, EC 3.2.1.35) has been isolated from pig liver and purified 1720-fold with an overall yield of 9.5%. The enzyme was purified using an acid-extraction technique followed by successive chromatography on DEAE-cellulose, two boronate affinity columns and Sephadex G-75. This final preparation, which was essentially homogenous as determined by gel electrophoresis, was a single subunit enzyme of apparent molecular weight 70000 with an isoelectric point of 5.0. No contaminant enzymes capable of degrading glycosaminoglycans could be detected in the final preparation. The substrate specificity of the enzyme was the same as for bovine testicular hyaluronidase; however, both the K m and l/ values were significantly lower for the pig liver enzyme with all of the substrates tested (hyaluronate, chondroitin 4-sulphate, chondroitin 6-sulphate). A full kinetic analysis of the enzyme using hyaluronate as a substrate showed that the activity of pig liver hyaluronidase was uncompetitively activated by either protons or NaCI.

Introduction Mammalian hyaluronidases (hyaluronate 4-glycanohydrolase, EC 3.2.1.35) are endoglycosaminidases that can hydrolyse the (ill ~ 4)glycosidic bonds in a number of different substrates such as hyaluronic acid, chondroitin 4-sulphate and chondroitin 6-sulphate. The most extensively studied form of the enzyme has been that obtained from bovine testes and several papers have been published outlining its purification and properties (see for example Refs. 1 and 2). Recently, interest has been shown in the clinical use of bovine testicular hyaluronidase for the treatment of myocardial infarction and certain other clinical conditions. Clinical trials, currently in progress in the U.K. have shown that a highly purified preparation of bovine testicular hy* To whom correspondence should be addressed. Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulphonic acid. 0304-4165/85/$03.30 © 1985 Elsevier Science Publishers B.V.

aluronidase can significantly reduce the mortality rate of patients with myocardial infarction [3]. However, a potential problem in the clinical use of the enzyme may be the availability of sufficient raw materials. It is therefore essential that sources other than bovine testes are evaluated and that the properties of the purified enzymes obtained are compared to those of.bovine testicular hyaluronidase. Aronson and Davidson [4] have shown that rat liver contains significant quantities of a lysosomal hyaluronidase that has properties similar to those of the bovine testicular enzyme. More recently, there have been reports of lysosomal hyaluronidases in a range of species and tissues, although most of these sources of the enzyme could not be considered as suitable for large-scale enzyme preparation [4,5]. A survey of hyaluronidase activities in the liver of a number of vertebrates has indicated that pig liver might provide a suitable alternative source of the enzyme in terms of both

258 availability and total enzyme activity [6]. In the present paper, the purification of pig liver hyaluronidase by a combination of conventional and affinity-chromatography procedures is described, together with an evaluation of the properties of the enzyme. Materials and Methods

DEAE-cellulose (DE 52, Whatman Biochemicals, Maidstone, Kent, U.K.), Matrex gel phenyl boronate (Amicon Corp., Danvers, MA, U.S.A.) and Sephadex G-75 (superfine; Pharmacia, Uxbridge, U.K.) were prepared according to the manufacturers' instructions. Crude potassium hyaluronate (containing 2.3% protein and 25% chondroitin sulphate (manufacturer's code 36-241)) was used for the routine assay of hyaluronidase activity, whereas for other work a highly purified preparation of the substrate (containing over 95% potassium hyaluronate (manufacturer's code 36-242)) was utilized. These substrates, together with chondroitin 4-sulphate, chondroitin 6sulphate and dermatan sulphate, were purchased from Miles Laboratories, Stoke Poges, Bucks, U.K.). Phenolphthalein /~-D-glucuronide (Na +salt) and p-nitrophenol-2-acetamido-2-deoxy-fl-Dglucopyranoside were obtained from Koch-Light Laboratories, Colnbrook, Bucks., U.K. Dipotassium 2-hydroxy-5-nitrophenylsulphate (nitrocatechol sulphate) was a gift from Dr. F.A. Rose, Biochemistry Department, University College, Cardiff, U.K. Crude preparations of bovine testicular hyaluronidase (300 I.U/mg) were purchased from Miles Laboratories, and a sample of the highly purified form of that enzyme (40000 I.U./mg) was a gift from Biorex Laboratories, London, U.K. Pharmalyte carrier ampholytes were purchased from Pharmacia. All other chemicals, which were of the highest purity available, were purchased from either BDH Chemicals, Poole, Dorset, or Sigma (London) Chemical Co., Poole, Dorset, U.K. Measurement of hyaluronidase activity The activity of pig liver hyaluronidase was measured over a 90 min incubation period essentially by the method of Gacesa et al. [7]. Incubation mixtures (total vol. 0.5 ml.) contained 0.5 mg of potassium hyaluronate dissolved in 0.1 M sodium

citrate buffer (pH 4.15)/0.15 M NaCl and activity was expressed in terms of /~mol of reducing Nacetylhexosamine released per rain. The enzyme reaction was linear over the incubation time used in this assay; hydrolysis of the available hexosaminidic bonds would not exceed 3% under these conditions. To overcome the possibility of sequential degradation of the substrates by fl-glucuronidase and fl-N-acetylhexosaminidase a final concentration of 1.5 mM saccharo-l,4-1actone (an inhibitor of fl-glucuronidase) was used in the assay mixture for crude preparations of hyahironidase. Bovine testicular hyaluronidase was assayed at pH 4.0 using the method of Gacesa et al. [7]. To allow comparison with the results from other laboratories the activity of the bovine testicular enzyme was converted from /~mol N-acetylhexosamine released per rain to the International Unit after calibration of the assay procedure with the W.H.O. standard preparation of hyaluronidase [8]. Using the assay conditions described above it was estimated that one N-acetylhexosamine unit was equivalent to 16 700 I.U. of hyaluronidase. In kinetic studies on these enzymes the substrate concentration was varied over the range of 0.25 m g / m l to 2.0 m g / m l and constants were calculated from direct linear plots [9]. When enzyme activity was measured using either chondroitin 4-sulphate or dermatan sulphate as potential substrates the Park Johnson reducing sugar assay [10] was used instead of the standard methods. This was essential as N-acetylgalactosamine 4-sulphate does not form a chromophore with the Morgan-Elson reagent that is used in the conventional assay method. Measurement of contaminant enzyme activities At each stage in the purification procedure for pig liver hyaluronidase, the preparation was monitored for other enzymes capable of degrading glycosaminoglycans. In particular, fl-glucuronidase [11], fl-N-acetylhexosaminidase [11] and arylsulphatase [12] activities were measured using, respectively, phenolphthalein /~-D-glucuronide, p-nitrophenol, 2-acetamido-2-deoxy fl-D-glucopyranoside and nitrocatechol sulphate. Protein estimation For routine scanning of column eluents, protein

259 was determined by measuring the ultraviolet absorbance of solutions at 280 nm. Otherwise, protein concentration was determined with FolinCiocalteau reagent using bovine serum albumin (0.2-1.0 mg/ml) as a standard [13].

Polyacrylamide gel electrophoresis Pig liver hyaluronidase preparations were subjected to electrophoresis (4 mA/tube) on 7.5% (w/v) polyacrylamide gels using 0.035 M fl-alanine (pH 4.3) as a running buffer. Typically, 20/~g of protein was applied to each gel. Samples (typically, 20/xg) of the enzyme were also subjected to electrophoresis in the presence of sodium dodecyl sulphate and fl-mercaptoethanol. In both series of experiments protein was visualised after staining with Coomassie blue.

Isoelectric focussing Polyacrylamide gels were prepared by the method of Vesterberg [14]. Samples of the enzymes (10-20/tg) were applied to the gels and isoelectric focussing in the presence of Pharmalyte carrier ampholytes was performed on an LKB electrophoresis apparatus for 3 h at 30 W/gel. Immediately after the current had been switched off, the pH gradient across the gel was measured using a flathead electrode (ELL, Chertsey, Surrey, U.K.) and the protein visualized by staining with Coomassie blue. A kit of isoelectric point marker proteins (Pharmacia) was used to check that the system was working satisfactorily.

Purification of pig liver hyaluronidase All purification procedures were carried out at 0-4°C and enzyme samples were stored at - 20°C. Pig livers were removed from the animals immediately after killing, put on ice and were transported back to the laboratory where the extraction procedure commenced without delay. Stage 1. Pig liver (140 g) was homogenised in 500 ml of ice-cold water for 2 rain at maximum speed in a Waring blender. The homogenate was filtered through muslin and centrifuged at 16000 × g for 30 min. The pellet was discarded, and the supernatant, which contained the bulk of the enzyme activity, was retained for further treatment. Stage 2. The pig liver supernatant was adjusted to pH 3.5 by the addition of 2 M HC1. After

maintaining the supernatant at this pH for 30 min, the resulting precipitate was removed by centrifugation at 16000 × g for 25 min. The supernatant was adjusted to pH 6.5 with 2 M NaOH immediately after centrifugation and then used for stage 3. The crude enzyme preparation could be stored at - 2 0 ° C at this stage if required. Stage 3. Material from stage 2 (total vol. 285 ml) was loaded on to a column (2.5 × 50.0 cm) of DEAE-cellulose (DE 52) that had been equilibrated with 0.01 M sodium phosphate buffer (pH 8.0). The column was eluted with 0.6 1 of a linear gradient of (0.01-0.2 M) sodium phosphate buffer (pH 8.0). Fractions were collected and assayed for hyahironidase, fl-glucuronidase, fl-N-acetylhexosaminidase and arylsulphatase activities and for protein. The eluate containing the bulk of the hyaluronidase activity was pooled and dialyzed against 0.02 M Hepes buffer (pH 8.0)/10 mM MgCI2/10 mM CaC12. For stages 4 and 5 material from stage 3 (total vol. 143 ml) was divided into two equal parts which were then treated in identical fashion. Active material was recombined after stage 5. Stage 4. Fractionation was carried out using a column (0.9 x 14.0 cm) of Matrex gel phenyl boronate (PBA-60) that had been equilibrated with 0.02 M Hepes buffer (pH 8.0). Ehition of material was by the sequential addition of 0.02 M Hepes buffer (pH 8.0) and 0.02 M Hepes buffer (pH 8.0)/100 mM a-o-mannose. All of these buffers contained 10 mM MgCl 2 and 10 mM CaCl 2 in addition to the components already stated. Fractions containing hyaluronidase activity were pooled. Stage 5. Material from stage 4 was dialysed against 0.02 M Hepes buffer (pH 8.0), but in the absence of MgC12 or CaCI2, and was fractionated on a second column of Matrex gel phenyl boronate (PBA-60) that had been equilibrated with 0.02 M Hepes buffer (pH 8.0). The column was washed sequentially with the same buffers as described in stage 4, except that no MgCl 2 or CaC12 was present. Fractions containing enzyme activity were pooled. Stage 6. The stage 5 preparation (total vol. 16.5 ml) was dialysed against 0.1 M sodium citrate buffer (pH 4.15)/0.15 M NaCl and then reduced in volume by dialysis against dry Sephadex G-25

260

powder. The hyaluronidase sample (vol. 5.25 ml) was applied to a column of Sephadex G-75 superfine (0.9 x 60.0 cm) and eluted with 0.1 M sodium citrate buffer (pH 4.15)/0.15 M NaC1. The purified enzyme was stored at 4°C until required.

contains at least one high-mannose, N-linked oligosaccharide unit [16]. In the preparation of the pig liver enzyme, repeating the boronate affinity chromatography step in the absence of divalent cations enabled a further purification to be obtained; however, it is not clear why this should have been so. The final purified preparation of pig liver hyaluronidase migrated as a single band on polyacrylamide gel electrophoresis at pH 4.3 (Fig. 2a). Electrophoresis in the presence of SDS and /3mercaptoethanol produced one major band corresponding to a subunit apparent molecular weight of 70000 and trace amounts of two other bands (Fig. 2b). A similar molecular weight was obtained under non-dissociating conditions, indicating that pig liver hyaluronidase is a single subunit enzyme in common with a wide range of hyaluronidase preparations from mammalian sources [1,2,4]. Isoelectric focussing of the purified pig liver hyaluronidase produced a single protein band with a p l of 5.0; this compared with a pl of 8.6 that was obtained for the bovine testicular enzyme.

Results and Discussion

Enzyme purification A number of preliminary experiments were carried out using crude extracts of pig liver in order to determine suitable assay conditions for hyaluronidase. In the presence of 1 m g / m l potassium hyaluronate and 0.1 M citrate buffer/0.15 M NaC1, the enzyme exhibited maximum activity at pH 4.15. The rate of reaction of the enzyme was linear for at least 1.5 h. These conditions were subsequently used for routine enzyme assay. Overall, the purification of pig liver hyaluronidase was estimated to be in the region of 1700-fold, although it was difficult to measure enzyme activity with accuracy in stages prior to the acid treatment (Table I, Fig. 1). The total recovery of enzyme activity was approx. 10%, which is comparable to results obtained by other groups for lysosomal hyaluronidase preparations [4]. The successful use of affinity chromatography on boronate gels implied that pig liver hyaluronidase is a glycoprotein with a significant mannose content. Other hyaluronidases have also been shown to contain carbohydrate [1] and some have been successfully purified on Con A-Sepharose which is known to be specific for mannose/glucose residues [15]. Results from our laboratory have shown that bovine testicular hyaluronidase

Contaminating enzyme actiL, ities

The purified pig liver hyaluronidase preparation does not contain any other glycosaminoglycan-degrading enzymes (Table II). Ion-exchange chromatography on DEAE-cellulose was a particularly effective step for the removal of /3-N-acetyl hexosaminidase. It is interesting to note that affinity chromatography on boronate gels in the presence of cations was effective in removing only arylsulphatase B. However, when the procedure was repeated using the same buffer conditions but

TABLE I P U R I F I C A T I O N OF PIG LIVER H Y A L U R O N I D A S E Stage

Volume (ml)

Total protein (mg)

Total units

% recovery

Pig liver homogenate Pig liver supernatant Acid-treated supernatant Ion-exchange on DE52 Boronate gel I Boronate gel II Sephadex G-75

500.0 330.0 285.0 143.0 61.2 16.5 5.8

22 900 10 560 3 363 473 91 8.1 1.4

1.26 1.20 1.31 0.63 0.62 0.24 0.12

100.0 95.0 104.0 50.0 49.0 19.0 9.5

Spec. act. U / m g ( × 103 ) 0.05 0.11 0.39 1.33 6.8 29.0 86.0

Purification (-fold) 1 2 8 27 136 580 1 720

261

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HYALURONIDASE ACTIVITY IS IN nmol o m i n - 1 , ml -1

Fig. 1. (Left.) Purification of pig liver hyaluronidase. (a) Stage 3 - chromatography on DEAE-cellulose using a linear gradient of 0.01 M-0.2 M phosphate buffer (pH 8.0). (b) Stage 4 - chromatography on Matrex gel phenyl boronate (PBA-60) using 0.02 M Hepes buffer containing 10 m M MgCI~ and 10 mM CaCI 2. a-D-Mannose (100 mM) in buffer was used to elute the enzyme activity. (c) Stage 5 - chromatography on Matrex gel phenyl boronate (PBA-60). Details are the same as for Stage 4 except the MgCI 2 and CaCI 2 were omitted. (d) Stage 6 - gel filtration on Sephadex G-75 (superfine) using 0.1 M citrate buffer (pH 4.15) and 0.15 M NaC1 as eluant. Full details are in the text. Bars indicate the pooled fractions. Hyaluronidase activity (11) and protein concentrations (o) are indicated. Fig. 2. (Right.) Polyacrylamide gel electrophoresis of purified hyaluronidase. (a) Non-denaturing conditions (pH 4.3). (b) Denaturing conditions (SDS and/3-mercaptoethanol).

in the absence of cations, this method was able to remove selectively both fl-glucuronidase and fl-Nacetylhexosaminidase from the hyaluronidase preparation. This suggests that the selectivity of boronate gels may be improved by the omission of divalent cations from the eluting buffer.

Effect of pH and ionic strength on e n z y m e activity Kinetic constants were determined for the purified enzyme at a variety of pH values and at two different ionic strengths using potassium hyaluronate as substrate. Previous work in our laboratory [7] has shown that buffer ions can have a profound effect upon the pH optimum of bovine

testicular hyaluronidase. Therefore, to simplify the interpretation of the data for the pig liver enzyme, no buffers were used in the incubation mixture; no change in pH occurred during the reaction. Enzyme activity was determined in the presence of either 0.1 M or 0.3 M NaC1 and the pH of the incubation mixture was adjusted to the required value by the addition of trace quantities of HCI. At the lower ionic strength, a pH optimum of 4.0 was observed, whereas, at the higher ionic strength, a shift in pH optimum to 3.4 occurred together with an increase in maximum velocity (Fig. 3). Qualitatively, these results are similar to those that have been obtained for bovine testicular

262 TABLE II C O N T A M I N A T I N G E N Z Y M E ACTIVITIES Enzymes, potentially capable of degrading glycosaminoglycans, were monitored throughout the purification procedure. Specific activities are expressed as u n i t s / r a g ( × 109) where one unit of enzyme activity is defined as the production of 1/*tool/rain of product. The final specific activity of the purified hyaluronidase preparation was 8.6.10 2/,mol reducing N-acetylhexosamine p r o d u c e d / m i n per mg. Specific activities (units/mg)( × 10 9)

Purification stage

fl-glucuronidase

fl-N-acetylhexosaminidase

arylsulphatase A

arylsulphatase B

55 000 840 720 0 0

35.0 5.5 4.4 0 0

17.8 25.3 29.3 11.6 0

49.4 12.6 0 0 0

Acid treated DEAE-cellulose Boronate gel I Boronate gel I1 Sephadex G-75

hyaluronidase [17]. Further analysis of the kinetic constants reveals that the value of V / K m remains constant over the range of ionic strength and pH values studied. This is indicative of an uncompetitive activation mechanism as shown in Eqn. 1, in which either H + or Na + may act as an activator of enzyme activity: A E+S~

ES

~ EAS ~ Products

I

I

(1)

Substrate specificity The activity of the enzyme was tested against a range of potential glycosaminoglycan substrates. Kinetic constants were obtained using incubation conditions similar to those described in the preceding experiment when high ionic strength was used (0.3 M NaC1 (pH 3.4)). Results are tabulated (Table Ill) for three substrates, and data for pure

I

I

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I

I

I

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T A B L E IlI

lo0

SUBSTRATE SPECIFICITY ALURONIDASE

OF

PIG

LIVER

HY-

Enzyme activity was measured at pH 3.5 in the presence of 0.3 M NaC1, but in the absence of buffers. Kinetic constants were determined from initial rates measured at a m i n i m u m of five different substrate concentrations. The figures in parenthesis are the constants obtained using bovine testicular hyaluronidase. n.d., not determined.

'7 ~ o°5 E E

I

I

I

I

I

I

2-6 3*0 3"4 3-8 4-2 4'6 5"0 5-4 pH Fig. 3. Dependence of kinetic constants on pH. Enzyme activity was measured at each pH value in the presence of 0.1 M (11) and 0.3 M NaC1 (e) but in the absence of buffers.

Substrate

K,~ ( m g / m l )

V(~mol/min)

Hyaluronate Chondroitin 6-sulphate Chondroitin 4-sulphate

0.64 (17.0) 0.28 (0.8) 0.34 (n.d.)

4.0.10 - 4 (5.25) 3.3.10-4 (0.1) 1.5-10- 4 (n.d.)

263

bovine testicular hyaluronidase are included in parentheses. The substrate specificity of both enzymes is qualitatively similar in that they are able to degrade hyaluronic acid, chondroitin 6-sulphate and chondroitin 4-sulphate. In general, the pig liver enzyme has lower values of K m and V than those obtained for bovine testicular hyaluronidase. Both enzymes also exhibited trace amounts of activity towards a commercial preparation of dermatan sulphate; however, this was not considered to be significant. It is well established that some commercial preparations of dermatan sulphate contain significant amounts of chondroitin 4sulphate-like regions that are degradable by hyaluronidases [18]. The general similarity between the pig liver enzyme and that from bovine testes indicates that the former may also have potential for use in clinical circumstances. Although attempts to scaleup the method have not yet been made, there seems no reason to suppose that insurmountable difficulties would be encountered.

Acknowledgements We wish to thank the SERC and Biorex Laboratories for financial support. M.B.J. held an SERC CASE award in association with Biorex Laboratories.

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