The distribution of 6- and 4-sulfated disaccharides in hybrid chondroitin SO4 chains synthesized by chondrocytes during endochondral bone formation will be determined. Chondromucoproteins will be isolated from monolayer cultures of chick embryo tibial chondrocytes and converted to chondroitin SO4 chains with (3H)xylitol residues at their reducing terminals by treatment with alkaline NaB3H4. Partial digestion of these labeled polymers with a mixture of hyaluronidase and excess Beta-glucuronidase will yield a mixture of end-labeled oligosaccharides having either 6- or 4-sulfated Ga1NAc residues at their nonreducing terminals. The polyacrylamide gel electrophoretic profiles of this mixture, observed by fluorography, will be compared with profiles of samples of the same mixtures that have been treated with either a 6-sulfatase or a 4-sulfatasy. The band displacements resulting from the specific sulfatase treatments will indicate the degrees of 6- and 4-sulfation occurring at increasing distances from the reducing terminal (3H)xylitol. Differential rates of hyaluronidase cleavage of 6- and 4-sulfated chondroitin SO4 sequences, observed in preliminary studies, will be optimized and used in the hyaluronidase cleavage step to obtain an indication of the occurrence of blocks of 6- or 4-sulfated disaccharides in the hybrid chains.