Hyaluronidase is a glycoprotein containing 5% mannose and 2,17% glucosamine, it catalyzes the random hydrolysis of 1,4-linkages between 2-acetamido- 2-deoxy- b-D-glucose and D-glucose residues in hyaluronate. It is a tetramer consisting of 4 equal subunits with a molecular mass of 14 kDa each.

• Enzyme Commission Number E.C. 3.2.1.35
• Extinction Coefficient (E1%): ~8
• Inhibitors: Fe²⁺, Fe³⁺, Mn²⁺ and Cu²⁺
• Sodium chloride acts as a stabilizer
• Dissolve 10 mg hyaluronic acid in 25 ml 0,1 M sodium phosphate buffer: pH 5,3 with 0,15 M sodium chloride

It degrades hyaluronan and has been found to be inappropriately regulated during cancer progression. These enzymes randomly cleave β-N-acetylhexosamine-[1→4] glycosidic bonds in hyaluronic acid, chondroitin, and chondroitin sulphates.

Testicular hyaluronidase hydrolyses the endo-N-acetylhexosaminic bonds of hyaluronic acid and chondroitin sulphuric acids A and C (but not B), primarily to tetrasaccharide residues. Monosaccharides are not liberated.

It is often used in conjunction with collagenase to dissociate the extracellular matrix between cells of animal tissue, in order to release viable cells for use in tissue culture. It may also be used to clarify synovial fluids in order to make cell counts possible.

Unit definition: Amount of enzyme which liberates one micromole of N-acetylglucosamine per minute at 37 °C and pH 4,0.