Hyaluronidase in Ocular Tissues* 1. Sensitive Biological Assay for Small Concentrations of Hyaluronidase

(1) The spreading reaction is a biological method, measuring the decomposing effect of hyaluronidase on the mucoids of the cement substance of rabbit's skin (Duran-Reynals, 1929). These substances consist partly of hyaluronic acid. Hyaluronidase increases the permeability of the tissues, and this involves an increased spreading capacity of liquids and corpuscular substances. This test is not specific for hyaluronidase as some unspecific agents also give a spreading reaction. (2) The viscosity-reducing method is based on the fact that hyaluronic acid is a substance of high viscosity (Madinaveitia and Quibell, 1940). The reduction of the viscosity caused by hyaluronidase is measured by this method.' (3) The turbidimetric method is most extensively used (Kass and Seastone, 1944). It is based on the observation that pure hyaluronate at pH 4-2 forms a stable turbid colloidal suspension with dilute serum, whereas depolymerized hyaluronate remains clear in these conditions. (4) Mucin clot prevention depends on the observation that native hyaluronic acid in acid solution coprecipitates with protein in a fibrous " mucin " clot (Robertson, Ropes, and Bauer, 1940). After incubation with hyaluronidase, hyaluronic acid becomes depolymerized and the mucin clot formation is prevented. The mechanism of this test is not clear. (5) The chemical method consists in measuring the increase in reducing sugar, or in liberated acetyl-glucosamine after depolymerization of hyaluronic acid.