The chemistry of connective tissues. 6. The constitution of the chondroitin sulphate–protein complex in cartilage

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Abstract
The polysac-charide chain weight (Cn), estimated by measuring the reducing power of the terminal residue, was compared with the osmotic molecular weight (Mn) for chondroitin sulphate-protein complexes extracted from bovine hyaline cartilage. Water extracts prepared from fresh cartilage slices in a high-speed homogenizer yielded a chondroitin sulphate-protein complex containing approximately 75% of chondroitin sulphate having Mn 1-5 x 106 and Cn 50 x 103. Thus the "osmotic molecule" contained 20-100 chondroitin sulphate chains terminating in reducing residues. Removal of protein by digestion with papain decreased Mn to 28 x 103 [plus or minus] 2000 and Cn remained unchanged. The result indicates that the chondroitin sulphate chains in the complex are linked together by protein and that the reducing terminals of a proportion of the chondroitin sulphate chains freed by papain are masked. When the native complex was treated with ovine hyaluronidase and the chondroitin sulphate chain fragments were removed by ultrafiltration, the chondroitin sulphate content of the remaining core was decreased to about 30% and Cn was 7730. The protein-rich core had Mn 170 x 103, showing the presence of 23 chondroitin sulphate chains of decreased length. The complex had approximately the same reducing power before as after treatment with hyaluronidase and ultrafiltration, thus showing that each chondroitin sulphate chain is attached to protein at one point only. It is concluded that the complex may exist in solution as a macromolecule of Mn 1-5 x 106 or an aggregate of smaller molecular units of Mn at least 750 x 103. The units contain at least 23 chondroitin sulphate chains of Cn 28 x 103. These are connected to a protein core by a single linkage which does not involve the terminal reducing residue.