Isolation and Purification of Bovine Testicular Hyaluronidase

We developed a one-step method for the chromatographic purification of hyaluronidase extract on Sepharose Blue, which produced the purified enzyme with a high yield (95%). Purification of hyaluronidase allowed us to obtain biological preparations standardized in protein composition and enzyme activity, which are useful for basic and applied studies.     

Resistance of Dextran-Modified Hyaluronidase to Inhibition by Heparin

Properties of native and aldehyde dextran-modified hyaluronidase (with surface amino group modification about 98%) were investigated. Optimal endoglycosidase activity of the native enzyme was observed at 0.15 M NaCl and pH 5.5 and electrostatic interactions influenced the enzyme activity. The inhibitory effect of heparin on hyaluronidase activity slightly differed at pH 5.5 (1.5-fold inhibition) and 7.5 (1.2-fold inhibition). Ionic strength of the reaction medium only slightly influenced the effect of heparin. Modification of hyaluronidase with dextran increased hydrophobic interactions and steric hindrance. Conjugation with dextran increased the resistance of hyaluronidase activity to denaturing agents (urea, guanidinium hydrobromide) and extended the optimal conditions for maximal endoglycosidase activity (pH 4.5-6.5, the range of NaCl concentration from 0.1 to 0.3 M). The conjugation also reduced electrostatic effects on the active site of hyaluronidase and efficacy of heparin inhibition. At pH 7.5 the enzyme was almost insensitive to heparin. The resistance of dextran-modified hyaluronidase to heparin points to approaches for subsequent studies of the heparin binding site of this enzyme and biomedical trial of the stabilized enzyme for the treatment of acute cardiovascular lesions.     

Chick embryo fibroblasts produce two forms of hyaluronidase

Cultured chick embryo fibroblasts derived from skin and skeletal muscle exhibit hyaluronidase activity both associated with the cell layer and secreted into the medium. Although both forms of the enzyme have a number of similar characteristics (R.W. Orkin and B.P. Toole, 1980, J. Biol. CHem. 255), they differ in thermal stability at neutral pH and in behavior on ion-exchange chromatography. Both forms of the enzyme are equally stable at acidic pH for long intervals, but the cell-associated hyaluronidase is significantly less stable than the secreted froms at neutral pH and at temperatures more than or equal to 30 degrees C. Neither the presence of proteases nor inhibitors of hyaluronidase appear to be involved in the cell-asspcoated enzyme. Chromatography of the two forms of hyaluronidase on carboxymethyl cellulose reveals that most (60-90 percent) of the secreted form of the enzyme elutes at a lower ionic strength than the cell- associated enzyme. Treatment of the secreted form of hyaluronidase with neuraminidase shifts its elution profile on carboxymethyl cellulose toward that of the cell-associated form, and also decreases its thermal stability at neutral pH. In contrast, treatment of the secreted form of hyaluronidase with alkaline phosphatase has no detectable effect. These data suggest that the secreted hyaluronidase differs from the cellular form in possessing additional sialic acid residues which endow the former with increased stability in the extracellular milieu.     

Purification and partial characterization of a novel hyaluronic acid-degrading enzyme from Antarctic krill (Euphausia superba)

Summary A novel enzyme degrading hyaluronic acid has been isolated, purified and characterized from Antarctic krill (Euphausia superba). A combination of affinity chromatography (Con A-Sepharose), gel filtration (Superose 6) and fast protein liquid chromatography (Mono Q) was used for the purification. The hyaluronidase activity was determined by a radial diffusion method based on hyaluronic acid incorporated into an agarose gel. Moreover, the beta-glucuronidase and endo-(1,3)-beta-D-glucanase activities were also followed through the process using phenolphtalein mono beta-glucuronic acid and laminarin as substrates. After the final purification step on Mono Q column, the chromatogram showed three main peaks designated A, B and C. Peak C contained high hyaluronidase activity undetectable in peak A and B. The betaglucuronidase activity was associated with peak A, while the endo-(1,3)-beta-D-glucanase activity was found in peak B and slight in peak C. The hyaluronidase was purified about 85-fold. It had a pH optimum of 5.3, a temperature optimum of 37°C and a molecular weight of 80 000 Daltons. On polyacrylamide gradient gel electrophoresis the enzyme fraction showed one major band associated with hyaluronic acid decomposition, slightly contaminated with a few other components. Isoelectric focusing in combination with a hyaluronic acid zymogram demonstrated one major band at pH 6.7 with high enzyme activity. Preliminary data on enzyme specificity suggest that krill hyaluronidase is a new endo-beta-glucuronidase and support the concept of krill enzymes as a remarkable and unusually effective digestive system adapted to the Antarctic marine ecosystem.     

Stimulation of bull sperm hyaluronidase by polycations.

The activity of bull sperm hyaluronidase (hyaluronate 3-glycanohydrolase, EC 3.2.1.36) is increased by the inclusion of polycations in the assay mixture. At pH 3.8, bovine serum albumin and histone give the greatest stimulation, while protamine sulfate, spermine, spermidine and hyamine 2389 stimulate to a lesser extent. Enzyme activity increases with serum albumin concentration to a nearly constant, high level at serum albumin concentrations greater than 1 mg/ml. Other stimulatory compounds show a similar concentration dependence except that inhibition of enzyme activity occurs at high concentrations of stimulator. The degree of stimulation depends on the pH, sample concentration and substrate concentration. Enzyme preparations with a low protein content give the greatest stimulation, while preparations with a high protein content show little stimulation. The concentration of serum albumin required for maximum stimulation increases with increased hyaluronic acid concentration. The results suggest that the stimulation of sperm hyaluronidase is nonspecific and results from an interaction of the polycation with hyaluronic acid. Since protein in the enzyme preparation substitutes for exogenous stimulator to a varying degree, serum albumin should be included in the assay mixture for sperm and testicular hyaluronidase to assure measurement of maximum enzyme activity.     

A developmentally regulated hyaluronidase of Haemonchus contortus

The trichostrongylid nematode Haemonchus contortus released a hyaluronic acid-degrading enzyme during in vitro development from the third (L3) to fourth (L4) larval stage. The enzyme did not degrade chondroitin sulfate A. Enzyme activity was optimal between pH 4.0 and 6.0, and the enzyme was inhibited by high concentrations of NaCl; the divalent cations Cu2+, Zn2+, Ca2+, and Mn2+ were not inhibitory. The hyaluronidase had a molecular mass estimated at 57 kDa by sucrose density gradient centrifugation and at 111 kDa by substrate sodium dodecyl sulfate polyacrylamide gel electrophoresis (reducing and nonreducing conditions), suggesting the formation of a dimer during the electrophoretic separation conditions. The level of hyaluronidase released during in vitro development peaked between 24 and 48 hr in culture and then gradually decreased, with little or no activity present in the 168-hr culture fluid. The enzyme was not detected in culture fluid from 24-hr incubations of either the mid-L4 stage (obtained from sheep 7 days postinfection) or the adult stage (obtained from sheep 30-35 days postinfection). The temporal expression of the hyaluronidase suggested a role for this enzyme in the early stages of the L3-L4 developmental process.     

A study of pathogenic factors of Streptococcus pneumoniae strains causing meningitis

Abstract Pneumococcal meningitis in St. Petersburg in the period 1985–1991 occurred in 1.7–2.3 children per 100 000 annually. The most common serotypes among pneumococcal strains isolated from patients with meningitis were 19, 1, 6, 15, and 2, whereas, among the capsulated strains isolated from carriers, type 3 predominated. Only one third of strains from cases of meningitis were highly virulent for mice (types 1, 2, 3). Hyaluronidase was produced by all the 39 studied strains, 22 (84.6±7.1%) out of 26 strains from patients with otitis media, and only by 15 (11.5±2.8%) out of 130 strains isolated from carriers. Non-capsulated strains lacked this enzyme. Results of intranasal inoculation of pneumococcal strains with different hyaluronidase activity and addition of exogenous hyaluronidase to strains which did not produce the enzyme confirm the hypothesis that this enzyme plays an important role in bacterial dissemination and breaching of the blood brain barrier by pneumococci. It was concluded that high hyaluronidase activity, presence of capsule, and pneumolysin or serotype (1, 2, and 19) despite hyaluronidase titer, are the most important factors contributing to the development of pneumococcal meningitis. The role of the mouse toxic factor is unclear.     

Improved Yield of High Molecular Weight Hyaluronic Acid Production in a Stable Strain of <Emphasis Type="Italic">Streptococcus zooepidemicus</Emphasis> via the Elimination of the Hyaluronidase-Encoding Gene

Despite the significant potential of Streptococcus zooepidemicus for hyaluronic acid (HA) production with high molecular weight (MW), the HA degrading properties of hyaluronidase prevents the bacteria to achieve enhanced HA yield with high MW. In the present study, we aim to knockout the hyaluronidase enzyme and assess its effects on the yield and MW of the produced HA. The kanamycin resistance gene between the left and right arms of hyaluronidase gene was inserted into pUC18 plasmid to construct pUC18-L-kana^r-R as a recombinant suicide plasmid. The construct was then transferred into S. zooepidemicus to induce the homologous recombination between the hyaluronidase gene and the kanamycin resistance gene. Gene deletion was confirmed by PCR and enzyme assay. The product was cultured on selectable medium in which the MW of HA was increased from 1.5 to 3.8 MDa. The yield of HA production using the mutant strain was higher in all different concentrations of glucose from 40 to 120 g/l. Moreover, glucose increase results in higher HA production within both wild-type and recombinant strains. However, the growth rate of HA concentration (the slope of the plot), as a consequence of increased glucose concentration, is always higher for the recombinant strain. Unlike the wild-type strain, there was no sharp HA production drop approaching the 6 g/l HA concentration. In conclusion, hyaluronidase activity and HA concentration and MW exhibited a mutual control on each other. Based on our results, deletion of the hyaluronidase gene positively affects the yield and MW of HA.     

Inhibition of hyaluronidase by fully O-sulfonated glycosaminoglycans.

We report a new flow injection assay (FIA) method for determining hyaluronidase activity and the inhibitory effects of chemical fully O-sulfonated glycosaminoglycans on this enzyme. The products of enzymatic action on hyaluronidase can be detected by FIA using fluorometric detection with the fluorogenic reagent 2-cyanoacetamide. The major products derived from hyaluronan by the action of mammalian testicular hyaluronidase (a hydrolyase) were confirmed by (1)H NMR spectroscopy and capillary electrophoresis. The FIA method was next applied to the assay of hyman urinary hyaluronidase activity and the screening of hyaluronidase inhibitors. The human urinary hyaluronidase activity measured ranged from 46 to 59 turbidity reducing units/mg protein. Among the glycosaminoglycans only heparin showed hyaluronidase inhibition. Chemically O-sulfonated glycosaminoglycans showed IC(50) values of hyaluronidase inhibition that correlated with the degree of O-sulfonation. Heparin was found to inhibit hyaluronidase activity noncompetitively, while chemically O-sulfonated HA strongly inhibited hyaluronidase through both competitive and noncompetitive effects.     

Activity of native and dextran-modified hyaluronidase in in vitro and in vivo systems

Modified hyaluronidase derivatives have been obtained. Covalent coupling of the enzyme with aldehyde dextran results in 65-85% protein binding to the carrier, residual catalytic activity accounting for 90-100% of the baseline. Modified hyaluronidase is more thermostable than the native enzyme. The data on intravenous drug distribution in the mouse organs are promising and ensure effective use of modified hyaluronidase for the treatment of pulmonary diseases.     

Interfacial behaviour of bovine testis hyaluronidase

The interfacial properties of bovine testicular hyaluronidase were investigated by demonstrating the association of hyaluronidase activity with membranes prepared from bovine testis. Protein adsorption to the air/water interface was investigated using surface pressure-area isotherms. In whichever way the interfacial films were obtained (protein injection or deposition), the hyaluronidase exhibited a significant affinity for the air/water interface. The isotherm obtained 180 min after protein injection into a pH 5.3 subphase was similar to the isotherm obtained after spreading the same amount of protein onto the same subphase, indicating that bovine testicular hyaluronidase molecules adopted a similar arrangement and/or conformation at the interface. Increasing the subphase pH from 5.3 to 8 resulted in changes of the protein isotherms. These modifications, which could correspond to the small pH-induced conformational changes observed by Fourier-transform IR spectroscopy, were discussed in relation to the pH influence on the hyaluronidase activity. Adding hyaluronic acid, the enzyme substrate, to the subphase tested the stability of the interfacial properties of hyaluronidase. The presence of hyaluronic acid in the subphase did not modify the protein adsorption and allowed substrate binding to a preformed film of hyaluronidase at pH 5.3, the optimal pH for the enzyme activity. Such effects of hyaluronic acid were not observed when the subphase was constituted of pure water, a medium where the enzyme activity was negligible. These influences of hyaluronic acid were discussed in relation to the modelled structure of bovine testis hyaluronidase where a hydrophobic region was proposed to be opposite of the catalytic site.     

Purification of bull sperm hyaluronidase by concanavalin-A affinity chromatography.

A new method for obtaining highly purified hyaluronidase (hyaluronate glycanohydrolase EC 3.2.1.25) in high yield is described. Bull seminal plasma was fractionated with (NH4)2 SO4 and the 30 to 65% saturation fractions were applied to a DEAE-cellulose column. The first protein peak contained hyaluronidase, beta-N-acetylglucosaminidase and beta-glucuronidase. The latter two enzymes were separated by gel filtration on Sephadex G-200. The hyaluronidase was further purified by a Concanavalin-A Sepharose 4B affinity column. By gradient elution with alpha-methyl-D-glucoside a fraction which had a specific activity of 1998 units/mg protein (57 942 National Formulary Standard units/mg protein) was obtained. The highly purified enzyme showed one major protein band on acrylamide gel electrophoresis at pH 4.3. The purified hyaluronidase did not show any beta-glucuronidase or beta-N-acetylglucosaminidase activities. The percent yield of purified hyaluronidase calculated on the basis of total activity was ten times higher than by any pervious method [Yang, C.H. and Srivastava, P.N. (1975) J. Biol. Chem. 250, 79-83].     

Multiple forms of ram and bull sperm hyaluronidase revealed by using monoclonal antibodies

Two monoclonal anti-sperm hyaluronidase-producing cell lines were isolated following inoculation of mice with ram sperm hyaluronidase monomer. Both lines produced antibodies of the IgG1 class; these bound to ram hyaluronidase after 'Western blotting' but did not recognize the native enzyme. Whereas the 1A4 antibody was specific for ram hyaluronidase, and did not react with 'blotted' bull, boar or rabbit hyaluronidase, the 1D6 antibody recognized bull as well as ram hyaluronidase. The antibodies could be used for immunocytochemical localization of hyaluronidase in fixed spermatozoa. However, although some form of denaturation was required to unmask or form the epitopes with which the antibodies reacted, the degree and type of fixation required was critical, for the epitopes were readily destroyed; in particular, they were very sensitive to chemical modification such as glutaraldehyde treatment. It could be demonstrated that, like ram, bull spermatozoa contained an extended oligomeric family of hyaluronidase forms, apparently the result of intermolecular disulphide cross-linking of monomers. In spermatozoa of both species, the enzyme was confined to the anterior acrosomal region of the head.     

Hyaluronidase in sera of tumour-bearing nude mice.

Cancer cell lines often secrete hyaluronidase, suggesting that this enzyme could be used as a marker of growing tumours. We have measured hyaluronidase in the sera of non-grafted mice and mice grafted with human tumour-derived hyaluronidase-secreting H460M and SA87 cells or non-secreting CB 193 cells. Mouse serum hyaluronidase was measured at pH 3.8 using the enzyme-linked sorbent assay (ELSA) technique by reference to human serum whose activity at pH 3.8 was determined by the Reissig technique. The serum hyaluronidase in non-grafted mice ranged from 310-520 mU l(-1) (mean+/-SD 432+/-70 mU l(-1), median 440 mU l(-1)). Hyaluronidase increased in the sera of tumour-bearing mice grafted with H460M cells or with SA87 cells, but not in the sera of mice grafted with CB 193 cells. Serum hyaluronidase activity in H460M or SA87 tumour-bearing mice correlated with the tumour mass, increased with time, and decreased after tumour removal. Zymography detected two different hyaluronidase forms in the sera of non-grafted mice: type 1 had only one hyaluronidase band and type 2 had five different bands. In both types, enzyme augmentation in tumour-bearing mice correlated with the presence of an additional enzyme band that was not seen in normal sera and that migrated as the cancer cell enzyme did; there was no augmentation of the normal isoform(s). These results show that serum hyaluronidase can be used to follow the development of tumours in mice grafted with hyaluronidase-secreting cells.     

Hyaluronidase in sera of tumour-bearing nude mice

Cancer cell lines often secrete hyaluronidase, suggesting that this enzyme could be used as a marker of growing tumours. We have measured hyaluronidase in the sera of non-grafted mice and mice grafted with human tumour-derived hyaluronidase-secreting H460M and SA87 cells or non-secreting CB 193 cells. Mouse serum hyaluronidase was measured at pH 3.8 using the enzyme-linked sorbent assay (ELSA) technique by reference to human serum whose activity at pH 3.8 was determined by the Reissig technique. The serum hyaluronidase in non-grafted mice ranged from 310-520 mU l^?1 (mean±SD 432±70 mU l^?1, median 440 mU l^?1). Hyaluronidase increased in the sera of tumour-bearing mice grafted with H460M cells or with SA87 cells, but not in the sera of mice grafted with CB 193 cells. Serum hyaluronidase activity in H460M or SA87 tumour-bearing mice correlated with the tumour mass, increased with time, and decreased after tumour removal. Zymography detected two different hyaluronidase forms in the sera of non-grafted mice: type 1 had only one hyaluronidase band and type 2 had five different bands. In both types, enzyme augmentation in tumour-bearing mice correlated with the presence of an additional enzyme band that was not seen in normal sera and that migrated as the cancer cell enzyme did; there was no augmentation of the normal isoform(s). These results show that serum hyaluronidase can be used to follow the development of tumours in mice grafted with hyaluronidase-secreting cells.     

Hyaluronidase in sera of tumour-bearing nude mice

Cancer cell lines often secrete hyaluronidase, suggesting that this enzyme could be used as a marker of growing tumours. We have measured hyaluronidase in the sera of non-grafted mice and mice grafted with human tumour-derived hyaluronidase-secreting H460M and SA87 cells or non-secreting CB 193 cells. Mouse serum hyaluronidase was measured at pH 3.8 using the enzyme-linked sorbent assay (ELSA) technique by reference to human serum whose activity at pH 3.8 was determined by the Reissig technique. The serum hyaluronidase in non-grafted mice ranged from 310-520 mU l^-1 (mean±SD 432±70 mU l^-1, median 440 mU l^-1). Hyaluronidase increased in the sera of tumour-bearing mice grafted with H460M cells or with SA87 cells, but not in the sera of mice grafted with CB 193 cells. Serum hyaluronidase activity in H460M or SA87 tumour-bearing mice correlated with the tumour mass, increased with time, and decreased after tumour removal. Zymography detected two different hyaluronidase forms in the sera of non-grafted mice: type 1 had only one hyaluronidase band and type 2 had five different bands. In both types, enzyme augmentation in tumour-bearing mice correlated with the presence of an additional enzyme band that was not seen in normal sera and that migrated as the cancer cell enzyme did; there was no augmentation of the normal isoform(s). These results show that serum hyaluronidase can be used to follow the development of tumours in mice grafted with hyaluronidase-secreting cells.     

Immunoenzymic studies on testicular hyaluronidase from rhesus monkeys (Macaca mulatta)

Hyaluronidase from rhesus monkey testes was purified by detergent extraction, ammonium sulphate fractionation, Sephadex G-200 column chromatography and concanavalin A-Sepharose affinity chromatography. The purified hyaluronidase showed one protein band on acrylamide gel electrophoresis. Antibodies to the purified hyaluronidase were raised in rabbits and showed a single precipitin line by Ouchterlony gel diffusion. The enzyme had a molecular weight of 62,000. The Km was 0.5 mg/ml for hydrolysis of hyaluronic acid at 37 degrees C. The optimum pH for the enzyme was 5.0 but activity was present over a broad pH range. The hyaluronidase was inhibited by HgCl2, CuSO4, FeSO4 and p-chloromercuribenzoate all at a concentration of 2 x 10(-4) M. Cysteine protected the enzyme against HgCl2 inhibition.     

Catalytic properties of testicular hyaluronidase after gamma-irradiation

The effect of gamma-irradiation on ovine testicular hyaluronidase was studied in aqueous solution. Following irradiation, hyaluronidase is inhibited, and the kinetics of inhibition follow a pattern in which Km and Vmax decline as radiation dose is increased. It was indicated that the binding affinity of the residual activity of hyaluronidase with substrate is enhanced and depends upon radiation damage. Effects of various agents such as pH, salts, PCMB and glutathione on irradiated hyaluronidase have been compared with non-irradiated enzyme. The irradiated hyaluronidase was more sensitive to inhibition by CuSO4 than the non-irradiated enzyme. The residual activity after irradiation is less refractory to FeCl3 inhibition and less sensitive to NaCl stimulation compared to non-irradiated hyaluronidase. pH response curves of ovine testicular hyaluronidase show two maxima which become more evident after irradiation.     

Do electrostatic interactions determine glycation of hyaluronidase derivatives with N-acetylhexosamines?

Using N-acetylglucosamine and N-acetylgalactosamine as model agents for glycation of native hyaluronidase and its chondroitin sulfate modified form it has been shown that the modified enzyme exhibited higher inactivation than the native enzyme, while heparin caused similar inhibition of both forms. Such effect could be attributed to the development of electrostatic interactions as the modified hyaluronidase had altered surface electrostatic potential after chondroitin sulfate binding. However, variations in ionic strength of the medium containing enzyme derivatives have shown that their endoglycosidase activity changed in a similar manner and the effect on glycation represents a multifactor process. N-acetylhexosamines are natural labels of endothelial glycocalyx degradation products. Interaction of the hyaluronidase forms with charged hyaluronan fragments revealed significantly higher inactivation of the modified enzyme compared with the native enzyme. The glycation pattern observed in this study was opposite to that observed with mono- and disaccharides. Thus, it appears that the investigated hyaluronidase derivatives represent an informative enzymatic test in vivo for determination of the dominant type of glycation agents in blood circulation and their origin.     

Development of a fluorescent substrate to measure hyaluronidase activity

A novel fluorescent substrate (termed FRET-HA) to quantitatively assess hyaluronidase activity was developed. Hyaluronan (HA), the major substrate for hyaluronidase, was dual labeled with fluorescein amine and rhodamine B amine. The fluorescein amine fluorescence signal was significantly quenched and the rhodamine B amine signal was significantly enhanced due to fluorescence resonance energy transfer (FRET). In the presence of bovine testes hyaluronidase, cleavage of HA disrupted FRET, resulting in a loss of the fluorescein amine quenching that was dependent on both enzyme concentration and time. Increase in the fluorescein amine signal could be conveniently monitored in both noncontinuous and continuous fashions. The K_m value for bovine testes hyaluronidase was determined using FRET-HA in a continuous fluorescent assay. Importantly, the estimated K_m value for bovine testes hyaluronidase using FRET-HA as the substrate was in excellent agreement with K_m values reported previously for this enzyme using native (i.e., unlabeled) HA. Therefore, FRET-HA is a reliable substrate for quantitatively assessing the HA/hyaluronidase molecular interaction. The simplicity, sensitivity, and versatility of the FRET-HA substrate suggest that it will have utility in a variety of assay platforms and should be a new tool for assessing hyaluronidase activity.