Hyaluronidase assay using a chondroitinsulphate substrate

Abstract

• 1.

  1. A spectrophotometric titration procedure for the estimation of chondroitin-sulphate using buffered toluidine blue has been developed. The titration readings are decreased by the action of hyaluronidase on chondroitinsulphate. Using this principle hyaluronidase has been assayed in amounts of 0.025–0.25 USP units. This range of activities corresponds with the depolymerisation of chondroitinsulphate at the rate of 45–450, μμequiv. glucuronic acid per min per 0.1 ml test solution.

• 2.

  2. The standard curves obtained with three different preparations of chondroitinsulphate differed little.

• 3.

  3. The assay is performed in the presence of 0.33 mM mercuric chloride. Ferric and ferrous salts at the same concentration inhibit the hyaluronidase activity. Crude serum albumin inhibits the activity slightly at the concentration present in blood.

• 4.

  4. The procedure has been shown to be applicable to the assay of hyaluronidase activity in fractions from blood serum and in serum itself.

• 5.

  5. The optimum pH of testicular hyaluronidase in the procedure and the nature of the products of the enzymic reaction have been studied.