Affinity of BAD-1 for heparin measured by SPR.

<p><b>(A)</b> BAD-1 binding to immobilized heparin monitored by surface plasmon resonance detection (SPR) using a Biorad Proteon XPR36. BAD-1 at the indicated concentrations was injected onto Biorad NLC neutravidin surface with biotinylated heparin immobilized to levels of 5 (circles) and 30 (squares) RUs. For clarity, only every 15th data point is shown. The solid lines are fits to the Langmuir binding model, on and off rates were fit to each sensogram but maximal response was fit to a single value for each immobilization level. The affinity was calculated from the rate constants to be 33±14 nM. <b>(B)</b> Heparin inhibition of BAD-1 binding to biotinylated heparin immobilized on a neutravidin surface. 0.375 µM BAD-1 with and without the addition of 3.75 µM heparin was injected onto the surface with heparin immobilized to levels of 30 (low), 59 (intermediate), and 96 (high) RU. Binding assays were performed using a buffer at physiological ionic strength (100 mM NaCl), similar to that in alveolar mucous (see Methods) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003464#ppat.1003464-Joris1" target="_blank">[16]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003464#ppat.1003464-Kozlova1" target="_blank">[17]</a>.