Hyaluronidase in Ocular Tissues : I. Sensitive Biological Assay for Small Concentrations of Hyaluronidase

SEVERAL methods for the estimation of hyaluronidase have been described:(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.None of the methods mentioned above is sensitive enough for the deter- mination of small quantities of hyaluronidase, for example those possibly present in the tissues of the eyes.The bacteriological decapsulation method is based on the observation that certain strains of mucoid streptococci of the Lancefield Groups A and C under definite conditions produce a capsule consisting of hyaluronic acid, which is dissolved in the presence of hyaluronidase (McClean, 1941a,b, 1942; Hirst, 1941).The bacteriological method is the most sensitive one but has been objected to (Meyer, 1947) because these organisms may lose their capsules under various conditions without added enzyme.