Panel b: The chemically O-sulfated S-link analog or natural O-link heparosan (starting materials, 0; ~0.5 μg/lane) were tested for cleavage by overnight treatment with either the recombinant human heparanase (H; 73 ng or 1.3 pmoles/lane) or a mixture of recombinant Flavobacterium heparin lyases I, II, and III (L; 0.017 units each/lane). Note: small sugar fragments do not fix/stain well in this system and run near the dye front.
Panel c: PAGE analysis of the kinetics of heparanase enzyme (Hepase; load 1 = 0.12 pmoles) cleavage of a fluorescent sulfo-O-linked heparosan substrate (load 1 = 3.3 pmoles; marked with arrow) with a titration of the S-linked analog inhibitor (either chemically or enzymatically sulfated polymer). The 20-min time point is shown in Fig. 4c; the 45-min samples are shown in Supplementary Fig. 55.
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