Effects of hyaluronan lyase, hyaluronidase, and chondroitin ABC lyase on mammalian vitreous gel

PURPOSE. TO determine the effects of enzymes on mammalian vitreous gel and to thus infer the structural roles of hyaluronan and chondroitin sulfate in the gel. METHODS. The wet weights of bovine vitreous gels were compared before and a fter incubation with Streptomyces hyaluronan lyase, chondroitin ABC lyase, testicular hyaluronidase, or buffer alone. The extent of hyaluronan depolym erization was determined by chromatography and that of chondroitin sulfate depolymerization by western blot analysis. RESULTS. After digestion with Streptomyces hyaluronan lyase (30 U/gel), the gel wet weight was the same as that of controls (incubated with buffer alo ne) despite 94% of the hyaluronan having been depolymerized; when digested with 100 U/gel, the gel wet weight decreased (to 57% of original wet weight versus 86% for controls, P = < 0.001) and hyaluronan was completely depoly merized. Chondroitin ABC lyase digestion (0.2 U/gel) resulted in a slight r eduction in gel wet weight (90% versus 96%, P = < 0.001) and depolymerizati on of 88% of the hyaluronan; the presence of fully digested chondroitin sul fate chains was established. Digestions with 100 and 500 U/gel of testicula r hyaluronidase resulted in a decrease (P = < 0.001, both cases) in gel wet weight (53% versus 82%, 100 U/gel; 57% versus 86%, 500 U/gel) with 75% and 97% hyaluronan depolymerization, respectively. CONCLUSIONS. Depolymerization of all vitreous hyaluronan and of chondroitin sulfate resulted in gel wet weight reduction but not gel destruction. Dige stion with 30 U/gel of Streptomyces hyaluronan lyase revealed a small pool (6%) of relatively enzyme-resistant hyaluronan that specifically contribute d toward maintaining gel wet weight.