Experimental details for Fig. 4: O-sulfo-S-linked heparosan analog enzyme challenges and inhibition

PH Peng He
XZ Xing Zhang
KX Ke Xia
DG Dixy E. Green
SB Sultan Baytas
YX Yongmei Xu
TP Truong Pham
JL Jian Liu
FZ Fuming Zhang
AA Andrew Almond
RL Robert J. Linhardt
PD Paul L. DeAngelis
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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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