## 2 Results
### An Increased Number of GAG-Binding Motifs Results in Higher Affinity for HS  
To assess the affinity of the peptides for different GAGs, IFT measurements were performed with HS, LMWH, dsLMWH and HA. The fluorescence of the conjugated fluorophore TAMRA at the NH\({}_{2}\)-terminus of the peptides was detected. First, IFT measurements were performed to evaluate the binding of the different TAMRA-labeled peptides to HS. The binding isotherms and their corresponding dissociation constant (K\({}_{\rm d}\)) values are shown in Figure 2A,B, respectively. The binding isotherms of the trimer peptides (KKAK-, KKQKASAS- and KKQK-3 mer) are characterized by steeper slopes in comparison with the binding isotherms of the dimer peptides (KKAK-, KKQKASAS-, KKQK- and KKKQQK-2 mer). Accordingly, the K\({}_{\rm d}\) values of the trimer peptides are lower than those of the dimer peptides, indicating higher affinity for HS. Furthermore, CXCL9(74-103) showed the highest affinity for HS. In view of this, peptide length and the number and type of GAG-binding motifs seem to contribute to HS binding affinity, as longer peptides with extra GAG-binding motifs tend to show higher affinity for HS. Additional IFT measurements were performed with LMWH, dsLMWH and HA for all TAMRA-labeled peptides. The binding isotherms and K\({}_{\rm d}\) values for LMWH are depicted in Figure 2C,D. In comparison with HS, the difference between dimer and trimer peptides is less pronounced on LMWH, especially for the KKAK-peptides. The affinity of the KKQK-3 mer is similar for HS and LMWH. Furthermore, the peptide with the highest affinity for LMWH is CXCL9(74-103). In addition, with dsDNA(Figure 2E,F), the highest affinity was measured for CXCL9(74-103). Remarkably, the peptide with the highest affinity for HA (Figure 2G,H) appeared to be the KKAK-3 mer and not CXCL9(74-103). Figure 1: Identification of the chemically synthesized TAMRA-KKQK-3 mer. The peptide was synthesized using Fmoc chemistry. After deprotection, RP-HPLC was used to purify the peptide and detection was performed using ion trap mass spectrometry. A mass spectrum of the peptide is shown with the intensity of the detected ions on the y-axis and the mass/charge (\(m/z\)) ratios on the x-axis. Deconvolution software (Bruker) was used to determine the relative molecular mass (M\({}_{\rm r}\)) of the uncharged peptide (insert). Figure 2: Peptides with more GAG-binding motifs have higher affinity for HS. Isothermal fluorescence titration (IFT) measurements were performed to study the interactions between TAMRA-labeled peptides and different GAGs. (**A**) Binding isotherms for the different peptides are displayed per GAG: (**A**) HS; (**C**) LMWH; (**E**) dsDNA; (**G**) HA. The change in fluorescence intensity (\(\Delta\)F) divided by the intrinsic fluorescence of the peptide (F\({}_{0}\)) is shown on the y-axis. \(\Delta\)F is calculated by subtracting F\({}_{0}\) from the fluorescence emission measured at a certain GAG concentration (**F**). The concentration of added GAG is shown on the x-axis. Data points represent mean values \(\pm\) SEM (\(n\) = 3). Dissociation constant (K\({}_{\rm d}\)) values (\(\pm\)SD) that are derived from the binding isotherms are visualized in bar plots per GAG: (**B**) HS; (**D**) LMWH; (**F**) dsDNA; (**H**) HA. Gln Residues in Both GAG-Binding Motifs and Linker Sequences Increase the Affinity of Trimer Peptides for LMWH, dsLMWH and HS, but Not for HA  
The IFT measurements of the three trimer peptides (KKAK-, KKOK- and KKQKASAS-3 mer) allowed us to evaluate the contribution of different amino acids to GAG-binding affinity. The binding isotherms and their corresponding K\({}_{\text{d}}\) values are shown in Figures 3A-C and 3D, respectively. The effect of the presence of hydrophobic (Ala) versus hydrophilic (Gln) residues in the GAG-binding motifs could be investigated by comparing the KKKAK-3 mer (Figure 3A) with the KKQKASAS-3 mer (Figure 3B).