Neutrophil-derived Hp stored in specific granules binds to Hb. (A) Nonreduced pooled subcellular β-fractions from neutrophils were incubated with Hb-Sepharose beads and Sepharose control beads. Subsequently, beads were precipitated, and Hb, control eluates (contr. eluate), and pooled subcellular β-fractions (granula control [gran. contr.]) were subjected to Western blot analysis using rabbit anti-human Hp antibody. The Western blot demonstrates the presence of highly glycosylated neutrophil-derived Hp in eluates from Hb-Sepharose beads but not from Sepharose control beads, indicating that Hp contained in specific granules binds to Hb. (B) SPR analysis demonstrating the binding curve of highly glycosylated neutrophil-derived (granula) Hp and plasma-derived Hp to a Hb BIAcore sensor chip. Response units (RUs) correspond to the response difference between Hp and control samples. SPR analysis demonstrates similar binding curves/kinetics and dissociation rate constants (K_{d granula}, 3.9 × 10^-4 s^-1; K_{d plasma},1.7 × 10^-4 s^-1) for both types of Hp.