./ 



238 IMMUNO-CATALYSIS 



charide to monosaccharide. At pH 7.0, however, with proper buffering 

 and salt concentration, a large proportion of the reducing sugar liber- 

 ated by this enzyme appeared to be monosaccharide. Meyer, et ah 

 (1940a) had already shown that their CI. welchii preparation liberated 

 91 per cent of the theoretical amount of the reducing sugar when 

 acting at pH 6.0 (optimal pH 5.8) determined under their salt and 

 buffer conditions. According to Meyer (1947), the dual nature of the 

 two glycosidic linkages in hyaluronic acid, one belonging to the 

 N-acetylglucosamine, the other to the glucuronic acid moiety, suggests 

 that the depolymerization and hydrolysis into monosaccharides requires 

 two enzymes. Thus it was observed that pneumococcal hyaluronidase 

 hydrolyzed the substrate to 100 per cent of the theoretical amount 

 whereas testicular hyaluronidase hydrolyzed the substrate to only 50 

 per cent. The addition of the pneumococcal hyaluronidase to the non- 

 hydrolyzed residue brought about complete hydrolysis, while the addi- 

 tion of fresh testicular enzyme had a negligible effect. 



Experimenting with testis, CL welchii and streptococcal culture 

 filtrates, Humphrey (1946) observed that at pH 6 to 7.0 they acted 

 in different ways upon hyaluronic acid, although all of them caused 

 hydrolysis with the liberation of reducing substances and substances 

 which were estimated for N-acetylglucosamine contents. When assayed 

 under identical conditions of salt concentration and pH, testis and CI. 

 welchii enzyme preparations showed so close relationship between their 

 power to reduce the viscosity of hyaluronic acid and to bring about its 

 hydrolysis to small molecules that it was thought very probable that 

 these two actions are brought about by the same mechanism. Hum- 

 phrey (1946a) suggested that hyaluronic acid contains, beside glyco- 

 sidic linkages between glucuronic acid and N-acetylglucosamine, pre- 

 formed ring compounds involving N-acetylglucosamine, or that the 

 enzymic hydrolytic products are peculiarly liable to form such com- 

 pounds. The fact that the testis enzyme with almost negligible, and 

 streptococcus enzymes with negligible )8-glucosaminidase activity can 

 hydrolyze hyaluronic acid in characteristic fashion, led Humphrey 

 (1946b) to conclude that such activity is not necessary for hyaluroni- 

 dase, and this was borne out by the failure of ;8-glucosaminidase prepa- 

 rations to show hyaluronidase effects. 



The mechanism of the action of hyaluronidase on hyaluronic acid is 

 still far from being clarified. However, the fact that the enzyme (testis) 



