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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 homogeneous as determined by gel electrophoresis, was a single subunit enzyme of apparent molecular weight 70 000 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 Km and V 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 NaCl.

Purified bovine testicular hyaluronidase (hyaluronate 4-glycanohydrolase, EC 3.2.1.35) was inactivated by butane-2,3-dione in either borate or Hepes buffer, pH 8.3. The presence of borate enhanced the inactivation process which followed pseudo-first-order kinetics with a calculated second-order rate constant of 13.54M-1 min-1. Using kinetic data it was estimated that the modification of 1 mol arginine per mol enzyme was sufficient for inactivation to occur, whereas amino acid analysis indicated that 4 mol arginine had been modified. The inactivation process was partially prevented by using either competitive inhibitors or substrates of the enzyme, thus indicating that the essential arginine residue is close to the active site of hyaluronidase. A full kinetic analysis of the enzyme with either hyaluronic acid or chondroitin 6-sulphate as substrate showed that the activity of hyaluronidase was uncompetitively activated by either protons or NaCl. The product obtained by reduction of the corboxyl groups of hyaluronic acid to the corresponding alcohol groups was a competitive inhibitor. The possibility that the microenvironment of hyaluronic acid was responsible for the observed kinetic effects of pH and ionic strength was dispelled. It is concluded that these data are compatible with a mechanism that involves anionic interaction between a carboxyl group on the substrate and an arginine residue on the enzyme.

The induction of myocardial infarction in rats by ligation of the left-anterior coronary artery was confirmed by measurement of increased plasma levels of creatine kinase, aspartate aminotransferase and lactate dehydrogenase. Using this model system it has been established that intravenous administration of 125I-labelled hyaluronidase to rats resulted in a preferential uptake of the enzyme by damaged myocardium as compared to normal heart tissue.

Studies on the effect of pH and ionic strength upon the activity of purified bovine testicular hyaluronidase have shown that the pH optimum for the hydrolysis of hyaluronic acid occurs at 5.2 in the presence of, and at 6.0 in the absence of NaCl. Hydrolytic activity towards various mucopolysaccharide and hyaluronate octasaccharide substrates was dependent upon the presence of strong electrolyte (LiCl, NaCl, KCl, CsCl, NaNO3 and Na2SO4), maximum activity being obtained at electrolyte strengths of 0.2. Identical weights of sulphated and unsulphated mucopolysaccharides were hydrolysed at similar rates under optimal conditions, except that double chains of chondroitin 4-sulphate were hydrolysed at twice the rate of the other polysaccharides. Hydrolytic activity towards hyaluronate hexasaccharide was favoured at pH values below 5.2 whereas transglycosylation activity was favoured at higher pH. Hyaluronate tetrasaccharide was neither a substrate for the hydrolytic or transglycosylation activity, nor was it an inhibitor of the enzymic hydrolysis of hyaluronic acid. No conformational change in hyaluronic acid was detected by CD-spectroscopy in the presence of varying concentrations of salt and the collective results suggest that the salt effect is exerted on the enzyme rather than on the substrate.

A highly purified commercial preparation of bovine testicular hyaluronidase (GL enzyme, Hyalosidase) was labelled with 125iodine without measurable loss of enzyme activity. The labelled preparation was administered intravenously into rats and the serum half-life of hyaluronidase was determined by measurement of both radioactivity and enzyme activity. The short half-life of the enzyme in plasma could not be accounted for by excretion in the urine and bile. Tissue distribution studies showed that the major site of uptake was the liver (59.7% of the recovered dpm). This rapid uptake by the liver could be reduced significantly by the pre-administration of yeast mannan or ovalbumin (a mannose-terminated glycoprotein). This suggests that the uptake of hyaluronidase by the liver is mediated by a mannose-specific receptor. Very little radioactivity was found in the heart (0.2% of the recovered dpm).

The most recently published method for the assay of testicular hyaluronidase preparations was based on the premise that the enzyme also exhibited carboxylesterase activity towards indoxyl acetate. Studies on the relative enzyme activities of various hyaluronidase preparations towards hyaluronate and indoxyl acetate, the relative stabilities towards pH, temperature and mechanical shaking and the behaviour towards a variety of inhibitors, showed that the activities towards the two substrates reflected the presence of at least two different enzyme systems in the preparations. Gel chromatography and polyacrylamide-gel-electrophoresis experiments confirmed these conclusions and the collective findings clearly establish that methods based on the use of indoxyl acetate cannot be employed to measure testicular hyaluronidase activity.