Protein-SDS and Protein-Heparin Interactions

Wt and Arg173Pro (0.2 mg/mL) proteins were incubated with 0.2 mM SDS (either at pH 7.4 or 5.0) for 48 h at 37°C. Complex formation was detected by the binding to ThT as described above. Protein binding of heparin was tested following incubation of 0.2 mg/mL proteins with heparin at a 2:1 heparin/protein molar ratio for 48 h at 37°C. In order to confirm whether binding to heparin results in the formation of high molecular weight complexes, the incubation mixtures were analyzed by PAGGE. In a separate experiment, the Arg173Pro variant was incubated at 0.5 mg/mL and pH 7.4 for 24 h at 37°C in the absence or in the presence of heparin, and morphology was compared by atomic force microscopy (AFM) as described previously [13 (link)]. All images were obtained at room temperature using a Multimode-Nanoscope V (Veeco, Santa Barbara, CA) operating in tapping mode with an etched silicon Probe model Arrow-NCR-50 Nano World (cantilever resonance frequency: 258 kHz, Force constant 42 N/m; tip radius 5–10 nm). Typical scan rates were 1–1.5 Hz.