Identification of blood plasma proteins using heparin-coated magnetic chitosan particles

Abstract

Heparin was immobilized on magnetic chitosan particles to be used as a tool for human plasma protein identification. Chitosan was magnetized by co-precipitation with Fe²⁺/Fe³⁺ (MAG-CH). Heparin was functionalized with carbodiimide and N-hydroxysuccinimide and covalently linked to MAG-CH (MAG-CH-hep). X-ray diffraction confirmed the presence of chitosan and Fe₃O₄ in MAG-CH. This particle exhibited superparamagnetism and size between 100-300 μm. Human plasma diluted with 10 mM phosphate buffer (pH 5.5) or 50 mM Tris-HCl buffer (pH 8.5) was incubated with MAG-CH-hep, and the proteins fixed were eluted with the same buffers containing increasing concentrations of NaCl. The proteins obtained were investigated by SDS-PAGE, LC/MS, and biological activity tests (PT, aPTT, and enzymatic chromogenic assay). Inhibitors of the serpin family, prothrombin, and human albumin were identified in this study. Therefore, MAG-CH-hep can be used to purify these proteins and presents the following advantages: low-cost synthesis, magnetic separation, ion-exchange purification, and reusability.

Keywords: Bioaffinity; Heparin; Ion-exchange; Magnetic beads; Plasma proteins; Prothrombin; Serpin.