T Yamagata

Micromethods have been developed for the measurement of as little as 3 µg of chondroitin sulfate A, B, or C in mixtures with other mucopolysaccharides by the use of chondroitinase-ABC, chondroitinase-AC, chondro-4-sulfatase, and chondro-6-sulfatase. With these methods, 35S-labeled chondroitin sulfates A, B, and C in a given mixture have been precisely and rapidly determined by measuring radioactivity alone. The methods have also been utilized to determine chondroitin sulfates A, B, and C in as little as 4 ml of normal urine.

Abstract 1. An enzyme, has been purified to apparent homogeneity from extracts of Proteus vulgaris, NCTC 4636, which was adapted on a medium containing chondroitin sulfate C. It has the following properties. (a) At pH 8, it degrades chondroitin sulfates A, B, and C at greater rates than chondroitin and hyaluronic acid. It does not attack keratosulfate, heparin, or heparitin sulfate. (b) It carries out an elimination reaction, yielding Δ4,5-unsaturated disaccharides. (c) In the crude extract it is accompanied by two different types of sulfatase, which are removed during purification. 2. The two sulfatases, chondro-4-sulfatase and chondro-6-sulfatase, have been separated from chondroitinase-ABC and from each other; both are required for the hydrolytic desulfation of chondroitinase products, Δ4,5-unsaturated disaccharide sulfates. They do not attack polymer chondroitin sulfates, hexa-, penta-, tetra-, or trisaccharides derived from chondroitin sulfates A and C by digestion with crude testicular hyaluronidase, or acetylgalactosamine 4-and 6-sulfates. One of these enzymes, chondro-4-sulfatase, catalyzes the conversion of Δ4,5-unsaturated disaccharide 4-sulfate (i.e. the product from the degradation of chondroitin sulfate A or B by chondroitinase-ABC) and its saturated analogue (i.e. acetylchondrosin 4-sulfate) to the corresponding nonsulfated disaccharides and inorganic sulfate, but does not attack Δ4,5-unsaturated disaccharide 6-sulfate (i.e. the product from the degradation of chondroitin sulfate C by chondroitinase-ABC) or its saturated analogue (i.e. acetylchondrosin 6-sulfate). In contrast, chondro-6-sulfatase carries out the desulfation of the disaccharide 6-sulfates and acetylgalactosamine 4,6-disulfate at position 6 while it does not attack the disaccharide 4-sulfate isomers. 3. Another type of chondroitinase, chondroitinase-AC, has been purified also to apparent homogeneity from extracts of Flavobacterium heparinum, ATCC 13125, which was adapted on a medium containing chondroitin sulfate C. Its properties have been compared with those of chondroitinase-ABC from P. vulgaris. (a) Unlike chondroitinase-ABC, it has no measurable activity with chondroitin sulfate B; like chondroitinase-ABC it carries out essentially the same reactions with chondroitin sulfates A and C, chondroitin, and hyaluronic acid. (b) In the crude extract it is accompanied by an enzyme similar to chondroitinase-ABC, an enzyme similar to chondro-4-sulfatase, and a glucuronidase which hydrolyzes the β-glucuronidic bond of unsaturated disaccharides but not the bond of saturated disaccharides. All these accompanying enzymes are removed during purification.