App note

Screening Competitors of Heparin-Binding Proteins

Hundreds of proteins, and many viruses, bind heparan sulfate on cell surfaces. A heparin surface on the P4SPR measures that binding and finds the compounds that block it.

SystemP4SPR

ApplicationGlycobiology, inhibitor screening

MoleculesHeparin, growth factor, sulfotransferase, small-molecule competitor

In short: Researchers at the University of Liverpool and Keele University built a heparin surface for the P4SPR and used it to study two heparin-binding proteins. In a competition assay, they identified which sulfate groups an enzyme recognizes, and showed that the drug suramin blocks its binding to heparin.

The need

Heparan sulfate, a sulfated sugar chain on every cell surface, binds more than 800 proteins: growth factors, enzymes, cytokines, and the attachment proteins of many viruses. Heparin is its laboratory stand-in. Knowing which proteins bind it, which sulfation patterns they prefer, and which compounds block the binding feeds directly into drug discovery and glycobiology.

How it works

Streptavidin is captured on a biotin surface, and heparin carrying a single biotin at one end is captured on the streptavidin, so every chain is anchored the same way. A protein that binds heparin gives a signal. In the competition format, the protein is premixed with a candidate competitor before injection: the less it binds the surface, the stronger the competitor (Figure 1). Three channels carry heparin and the fourth, streptavidin only, is the reference.

Heparin chains anchored on streptavidin; proteins binding the chains; proteins with a competitor in their binding site unable to bind — Figure 1. Heparin anchored on streptavidin; a heparin-binding protein attaches to it; premixed with a competitor, the protein no longer reaches the surface.

What the study showed

	Result
Growth factor (FGF2)	Clear binding to heparin, none to the streptavidin reference channel; full regeneration
Sulfation preference	A library of selectively desulfated heparins showed which sulfate groups a heparan sulfate sulfotransferase needs
Small-molecule competitor	Suramin blocked the enzyme's heparin binding, half-maximal at about 5 µM (Figure 2)
Surface life	Biotin monolayers kept 3 months; heparin surfaces stored for 10 days and reused over a week

Simulated competition curve: enzyme binding to heparin falls as suramin increases, half blocked at about 5 micromolar — Figure 2. A competition curve, simulated from the half-maximal concentration reported for suramin. Each point is one injection of enzyme premixed with competitor.

Full method and data: Su et al., PeerJ Analytical Chemistry 2022 (reference below).

Why the P4SPR fits

What the lab needs	What the P4SPR brings
Glycans anchored the same way every time	Biotin capture on streptavidin: single-point, oriented attachment
Compounds that bind a protein, not the surface	Competition format: the compound is in solution, the readout is the protein's binding
A built-in check for non-specific binding	Four channels, read in turn: three with heparin, one streptavidin reference
Many injections per surface	Salt and acid, or mild detergent, fully regenerate the heparin surface
Surfaces ready when needed	Biotin surfaces can be prepared in batches and stored

Beyond heparin

Any biotinylated glycan, peptide or nucleic acid can be captured the same way, and the competition format works for any target whose binding partner can sit on the surface. For lectins and glycan antigens, see our note on glycan and lectin assays.

What you need

Item	Source
P4SPR	Affinité Instruments
Glycan Sensor (SAMGLY-01), or Biotin Sensor (SAMBT-01) with the Streptavidin Kit (STRP-KIT)	Affinité Instruments
Glycan Analysis Kit (GLY-KIT) and Regeneration Kit (REG-KIT)	Affinité Instruments
Heparin biotinylated at its reducing end	Prepared as described in the reference, or commercial
Heparin-binding protein and candidate competitors	Your proteins and compounds

Reference

D. Su, Y. Li, E. A. Yates, M. A. Skidmore, M. A. Lima, D. G. Fernig, "Analysis of protein-heparin interactions using a portable SPR instrument," PeerJ Analytical Chemistry, vol. 4, e15, 2022. doi:10.7717/peerj-achem.15 (open access, CC BY 4.0).

Illustrations by Affinité Instruments. Figure 2 is simulated from the value reported in the reference above, not measured data.