Fig. (1) Serp-1 binds to heparin in vitro. In a pull-down experiment with heparin/sepharose beads (A), Serp-1 (100 ng) was incubated with 15 µl of heparin/sepharose beads (Amersham) for one hour at 37 °C. After Serp-1 bound to heparin beads, 100 µl of NaCl solution at different concentrations (0 M, 1 M, and 2 M) was added to elute Serp-1 from the heparin beads. The elution was performed at room temperature for 10 minutes. After elution, the Serp-1 heparin beads were loaded on SDS-PAGE gel for electrophoresis and followed by the immunoblot detection with the anti-Serp-1 monoclonal antibody. 100 ng Serp-1 positive control shown in lane 1. FPLC chromatograph analysis: native (B) and heat-inactivated (C) Serp-1 were loaded onto the heparin columns and eluted with a NaCl gradient. The absorbance at 280 nm was monitored. A representative elution curve is presented from three repeat experiments. X axis at bottom indicates running time of the column analysis. X axis at top indicates collecting fractions (1 ml/fraction). Y axis indicates absorbance at 280 nm. Amino acid sequence alignment (D). Human PAI-1 helix D heparin-binding domain (13 amino acid residues) aligns with the Serp-1 sequence by MacVector (version 3). The residue (Arg6) in Serp-1 has been mutated (boxed) to examine its significance of the heparin binding ability (see text). The identical amino acids in both sequences are dotted and the similar amino acids are starred underneath. 3-D serpin structure showing the relative positions between helix D (hD) and the reactive center loop (RCL) is adapted from Gettins (Chem Rev 2002)