PURIFICATION, CHARACTERIZATION AND SPECIFICITY OF CHONDROITIN LYASES AND GLYCURONIDASE FROM FLAVOBACTERIUM-HEPARINUM

The chondroitin lyases from Flavobacterium heparinum (Cytophaga hepari nia) have been widely used in depolymerization of glycosaminoglycan an d proteoglycan chondroitin sulphates. Oligosaccharide products derived from chondroitin sulphate can be further degraded by glycuronidases a nd sulphatases obtained from the same organism. There has been no repo rted purification of these enzymes to homogeneity nor is there any inf ormation on their physical and kinetic characteristics. The absence of pure enzymes has resulted in a lack of understanding of the optimal c onditions for their catalytic activity and their substrate specificity . This has limited the use of these enzymes as reagents for preparatio n of oligosaccharides for structure and activity studies. Reproducible schemes to purify a chondroitin AC lyase, a glycuronidase and chondro itin B lyase from Flavobacterium heparinum to apparent homogeneity are described. Chondroitin AC lyase (chondroitinase AC, EC 4.2.2.5), glyc uronidase [chondro-(1 --> 3)-glycuronidase, no EC number] and chondroi tin B lyase (chondroitinase B, no EC number) have M(r) values (assesse d by SDS/PAGE) of 74 000, 41 800 and 55 200 respectively, and isoelect ric points (determined by isoelectric focusing) of 8.85, 9.28 and 9.05 respectively. Chondroitin lyase AC and B contain pyroglutamic acid at their N-termini precluding their analysis by Edman degradation. Deblo cking with pyroglutamate aminopeptidase facilitated the determination of their N-terminal sequences. The kinetic properties of these enzymes have been determined as well as the optimum conditions for their cata lytic activity. The specificity of the glycouronidase, determined usin g 17 different disaccharide substrates, shows that it only acts on uns ulphated or 6-O-sulphated 1 --> 3 linkages. The chondroitin lyases are both endolytic enzymes, and oligosaccharide mapping shows their expec ted specificity towards the chondroitin and dermatan sulphate polymers .