FIGURE 7.

Recombinant proteins F1₆₈₃–F5₆₈₃ bind heparin. A, binding of recombinant protein to biotinylated heparin. Total binding (■) was determined by coating recombinant protein F1₆₈₃–F5₆₈₃ (10 μg ml⁻¹) to 96-well microtiter plates followed by binding with increasing concentrations of biotinylated heparin (x axis). Nonspecific binding (▴) was determined by binding with unlabeled heparin in a 50-fold excess to biotinylated heparin. Specific binding (◊) was determined by subtracting the nonspecific from the total binding. K_d values for all five recombinant proteins were determined from the specific binding data. B, inhibition of heparin binding by glycosaminoglycans. Recombinant protein (10 μg ml⁻¹) was coated to 96-well microtiter plates and bound with biotinylated heparin in the presence of an increasing excess of inhibitor (x axis). Inhibitors included unlabeled heparin (■), fucoidan (▴), chondroitin sulfate A (▾), chondroitin sulfate B (♦), and porcine type II mucin (●). C, nondenatured recombinant protein dot blots. Recombinant proteins were spotted onto nitrocellulose membrane, blocked, and incubated with 30 μg ml⁻¹ biotinylated heparin. Bound recombinant protein amounts (above blots) are listed in micrograms. D, denatured recombinant protein dot blots. Recombinant proteins were denatured by boiling in an SDS-containing reducing solution before spotting onto nitrocellulose membrane and incubation with heparin as with C. Bound recombinant protein amounts (above blots) are listed in micrograms.