The interactions of the non-specific inhibitor of hyaluronidase with hyaluronidase and proteolytic enzymes in vivo

1. Following intravenous administration of testes hyaluronidase in rabbits and dogs, there is a decrease in the level of the non-specific inhibitor of hyaluronidase in serum. 2. If a large amount of hyaluronidase is injected, the inhibitor level is reduced to zero and hyaluronidase may be present in serum for some time after the injection. The hyaluronidase activity of such samples of serum increases when the serum is incubated with papain. 3. Hyaluronidase activity is found in the livers of the injected animals in large amounts and this activity is increased considerably when the homogenate of this tissue is incubated with papain. 4. Intravenous administration of several proteases or venom produces a decrease in the serum inhibitor level. Intravenous administration of streptokinase produces such a decrease in rabbits but not in dogs. 5. There is a correlation between the depletion of the inhibitor from the serum and the occurrence of a slow, persistent depression of blood pressure upon administration of proteolytic enzymes.     

The asparagine-linked carbohydrate of honeybee venom hyaluronidase

Hyaluronidase from the venom of the honeybee (Apis mellifera) has been purified by gelpermeation and cation exchange chromatography. Its asparagine-linked carbohydrate chains were released from tryptic glycopeptides with N-glycosidase A and reductively aminated with 2-aminopyridine. Separation of the fluorescent derivatives by size-fractionation and reversed-phase HPLC afforded eighteen fractions which were analysed by two-dimensional HPLC mapping combined with exoglycosidase digestions. The bulk of the N-linked glycans of hyaluronidase consisted of small oligosaccharides (Man_1–3GlcNAc₂), most of which were either 1,3-monofucosylated or 1,3-(1,6-)difucosylated at the innermost GlcNAc residue. High-mannose type structures constituted the minor fractions, together making up about 5% of the oligosaccharide pool from hyaluronidase. Four fractions, making up 8% of the N-linked glycans, contained the terminal trisaccharide GalNAc1-4[Fuc1-3]GlcNAc1- in 1,2-linkage to the core 1,3-mannosyl residue. No evidence for the presence of O-glycans or sialic acids could be found.Abbreviations GalNAc N-acetylgalactosamine - GlcNAc N-acetylglucosamine - PA pyridylamino - PLA phospholipase A₂ - 2D-HPLC two-dimensional HPLC     

The detection of hyaluronidase on electrophoresis membranes

A method is described for the detection of hyaluronidase enzyme activity following electrophoresis on cellulose acetate membranes prior to elution of the enzyme from the support medium. It appears to be specific for endo-N-acetylhexosaminidase activity. It detects 0.005 of the least amount of enzyme detected by a miniaturized turbidity reduction test or by a standard viscosity reduction test.