Purification and partial characterization of testicular hyaluronidase.

A new method is presented for the purification of testicular hyaluronidase involving ion exchange chromatography followed by gel filtration on Sephadex G-75. A highly purified hyaluronidase preparation has been obtained which contains 45,000 National Formulary activity units per mg, dry weight, and which migrates as a single component on polyacrylamide gel electrophoresis at pH 4.3. The enzyme has been shown to be a glycoprotein containing 5.00% mannose and 2.17% glucosamine (expressed as IV-acetylglucosamine). The molecular weight of the purified hyaluronidase has been determined to be 61,000 by gel filtration methods. This value is in contrast to a molecular weight of 126,000 for the crude enzyme, as determined by similar techniques. This difference between the crude and purified enzyme could be due to association with a carrier protein in the crude testicular extract or to the existence of hyaluronidase as a dimer in the crude state. Only 76% of the weight of the purified hyaluronidase could be accounted for by its amino acid and carbohydrate composition.