A novel assay to probe heparin-peptide interactions using pentapeptide-stabilized gold nanoparticles

In this article, we present a novel assay to probe the interactions between heparin and heparin-binding peptides based on CALNN pentapeptide-stabilized gold nanoparticles. This assay relies on rapid aggregation of gold nanoparticles and dramatic retardation in the presence of a large excess of heparin due to the binding of peptides to heparin. Using this method, the dissociation constant (K(d)) and melting temperature (T(m)) of three different peptides against heparin were determined. The results from capillary electrophoresis demonstrated that K(d) values measured by this method were comparatively accurate. It was found that the peptide with the lowest K(d) did not have the highest T(m). Structural analysis by circular dichroism was performed to explain this phenomenon. A comparison with the results from affinity chromatography indicates that electrostatic interactions only are not the major determinant of the affinity between heparin and peptide, but other interactions such as hydrogen-bonding and hydrophobic interactions may play important roles in the overall interactions. This novel assay is inexpensive, label-free, and easy to implement in the laboratories, does not suffer precipitation of the heparin-peptide complex or their conformational changes caused by surface immobilization, and is expected to be a useful complement to other existing methods.