Interaction of HRPII with GAGs. (A) Binding of HRPII to immobilized heparin. HRPII in 10mM NaCl binding buffer in the absence of Zn²⁺ (top) or presence of 15μM Zn²⁺ (bottom) was applied to a heparin-agarose chromatography column. After washing with binding buffer, fractions were collected with successive applications of increasing amounts of NaCl. Note that, after fraction 5 for each panel, EDTA (1mM) was included in the buffers for the elution of fractions 6-10. Fractions were analyzed for HRPII by immune-dotblots using mAB2G12 as described in “Binding assay.” (B) Competition of various unlabeled GAGs with tritiated heparin for binding to HRPII. A fixed concentration of tritiated heparin was mixed with fixed amounts of HRPII and zinc acetate along with various amounts of unlabeled GAGs in a 25-μL reaction volume in 96-well plate format. Thereafter, reactions were incubated at 37°C for 1 hour before processing to determine the amount of tritiated heparin bound to HRPII as described in “Competition assays.” Data are mean ± SE for triplicate wells.