Purification and properties of bacterial chondroitinases and chondrosulfatases.

1. An enzyme, “chondroitinase-ABC,” 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 A4,5-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 chondroitinaseABC and from each other; both are required for the hydrolytic desulfation of chondroitinase products, A4, S-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 4and 6-sulfates. One of these enzymes, chondro-4-sulfatase, catalyzes the conversion of A4,5-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 A4,5-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 4,6disulfate 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-