Heparin-mimetic sulfated peptides with modulated affinities for heparin-binding peptides and growth factors

Abstract

Heterogeneity in the composition and in the polydispersity of heparin has motivated the development of homogeneous heparin mimics, and peptides of appropriate sequence and chemical function have therefore recently emerged as potential replacements for heparin in selected applications. Here, we report the assessment of the binding affinities of multiple sulfated peptides (SPs) for a set of heparin-binding peptides (HBPs) and for vascular endothelial growth factor isoform 165 (VEGF165); these binding partners have application in the selective immobilization of proteins and in hydrogel formation through non-covalent interactions. Sulfated peptides were produced via solid-phase methods, and their affinity for the HBPs and VEGF165 was assessed via affinity liquid chromatography (ALC), surface plasmon resonance (SPR), and in selected cases, isothermal titration calorimetry (ITC). The shortest peptide, SP(a), showed the highest affinity binding of HBPs and VEGF165 in both ALC and SPR measurements, with slight exceptions. Of the investigated HBPs, a peptide based on the heparin-binding domain of human platelet factor 4 showed greatest binding affinities toward all of the SPs, consistent with its stronger binding to heparin. The affinity between SP(a) and PF4(ZIP) was indicated via SPR (K(D)=5.27 microM) and confirmed via ITC (K(D)=8.09 microM). The binding by SP(a) of both VEGF and HBPs suggests its use as a binding partner to multiple species, and the use of these interactions in assembly of materials. Given that the peptide sequences can be varied to control binding affinity and selectivity, opportunities are also suggested for the production of a wider array of matrices with selective binding and release properties useful for biomaterials applications.

Fig. 1

The ¹H NMR (400 MHz) spectra of non-sulfated and sulfated tyrosines, and the sulfated tyrosine-containing peptide. (a) Fmoc-Tyr(OH)-OH: δ = 6.69 (2H, tyrosyl meta, d), 7.04 (2H, tyrosyl ortho, d). (b) Fmoc-Tyr(OSO₃)-OH: δ = 7.20 (4H, sulfated tyrosyl aromatic, m). (c) SP_a peptide: δ = 7.20 ppm (8H, sulfated tyrosyl aromatic, m).

Fig. 2

The affinity chromatography of sulfated peptides/heparin and heparin-binding peptides/VEGF165. The NaCl concentration required to elute the bound analytes from sulfated peptidyl or heparin columns was recorded as an indicator of binding affinity. The error bars were derived from standard deviations of the average of three or more samples, except for VEGF165. The chromatography data of heparin column and ATIII, HIP, and PF4 were taken from previous reports [66,69].

Fig. 3

The measured dissociation constants of sulfated peptides/heparin and their binding partners measured via SPR. A series of concentrations of ATIII, HIP, PF4, and VEGF165 analytes were injected to the sulfated peptides/heparin immobilized sensor chip. The binding responses with various concentrations of analytes were fit to a Langmuir model in order to give the binding constants. The insets show the K_D of SP_a and heparin in log-scale. The error bars are derived from the standard deviations of multiple samples. The data of heparin binding with ATIII, HIP, and PF4 were taken from previous reports [66,69].

Fig. 4

Binding isotherm of SP_a interacting with PF4_ZIP. The total heat of each injection was integrated and plotted (■). The solid line was the fit of the experimental data which was used to calculate K_D, ΔH, and ΔS.

Scheme 1

Non-covalently assembled hydrogels for growth factor delivery. PEG stands for poly(ethylene glycol); LMWH for low molecular weight heparin; HBP for heparin-binding peptide; GF for growth factor.