Differences between two fractions of glycosaminoglycans of the corneal stroma in their structural relation to collagen

Abstract

Glycosaminoglycans (GAG) of bovine cornea were sequentially extracted by 0.15 M NaCl and by 1 M CaCl2, pH 8. The amounts of hexosamine (HexN) and hexuronic acid (HexUA), specific hexoses (Hex), and protein were determined in the extracts. The ultrastructure of the corneal stroma after NaCl and NaCl/CaCl2 extraction was also studied. Approximately 70% of the Hex-HexN extractable by 0.15 M NaCl is removed in the first NaCl extract, with the amount decreasing rapidly to the fifth NaCl extract. Only 20-30% of the extractable HexUA-HexN is removed in the first NaCl extract; subsequent extraction removes successively less HexUA-HexN. There is a sharp increase, however, in both Hex-HexN and HexUA-HexN removed in the first 1 M CaCl2 extract, ranging from 25 to 40% of the total extractable with NaCl. The stroma retained lamellar organization and normal spacing, diameter, and length of collagen fibers after NaCl extraction. Visibility of the 64-nm periodicity was enhanced because of loss of density in the ground substance. CaCl2 extraction led to loss of lamellar organization and apparent disruption of the collagen fibers. Only short thin fibrils remained, embedded in a material having the density and very fine fibrillar organization of basal lamina. Disruption of the collagen fibers is probably due to removal of a specific GAG component necessary to maintain the collagen as an extended morphologic structure but may also be due to a specific degradative effect of CaCl2 on collagen no longer covered by a protective layer of GAG and glycoproteins.