A surface plasmon resonance-based solution affinity assay for heparan sulfate-binding proteins.

Area of Science:

• Biochemistry
• Analytical Chemistry

Background:

• Heparin and heparan sulfate-binding proteins play crucial roles in various biological processes.
• Accurate measurement of binding affinities (K(d)) is essential for understanding these interactions and for drug discovery.

Purpose of the Study:

• To develop and validate a surface plasmon resonance (SPR)-based solution affinity assay for quantifying the binding affinity of heparin/heparan sulfate-binding proteins to diverse ligands.
• To demonstrate the assay's applicability across a range of proteins and ligands.

Main Methods:

• Utilized a pre-equilibrated solution of protein and ligand passed over a heparin-immobilized sensor chip.
• Employed SPR to measure binding interactions.
• Compared heparin sensor chips prepared via biotin-streptavidin affinity capture and direct covalent attachment.

Main Results:

• Successfully measured K(d) values for heparin/heparan sulfate-binding proteins binding to various ligands.
• Achieved consistent K(d) values regardless of the heparin sensor chip preparation method.
• Demonstrated broad applicability to diverse proteins (e.g., FGF-1, VEGF, IL-8) and ligands (e.g., heparin, PI-88).

Conclusions:

• The developed SPR assay is a robust and versatile tool for measuring heparin-binding protein affinities.
• The assay is well-suited for high-throughput screening of ligands in drug discovery programs targeting heparin-mediated interactions.