Instead, a speciation model was developed assuming two sets of binding sites with different dissociation constants 68 to mimic high and low affinity binding sites, allowing the simulation of PDS modulation depths of HRG pseudo-titration samples in dependence of the Cu II loading (for a full description of the model and simulation parameters see SI and J. L. Wort et al., manuscript in preparation). Importantly, the model unambiguously demonstrated that experimental modulation depths could be simulated with 12 high affinity binding sites (KD << 1.25 × 10 -4 ), while observed plateauing of PDS data could not be satisfactorily described by the model if more high affinity sites were assumed (Figures and-19, Tables ). Note, however, that the model did not allow firm conclusions regarding the number or dissociation constant of low affinity binding sites. It should be emphasised that, while PDS (Figure ) yielded very broad distance distributions that we refrained from quantifying and that did not change significantly within confidence intervals between 1 and 20 equivalents of Cu II added, reliable modulation depth quantitation has been possible and facilitated robust spin counting, as recently benchmarked. In summary, the presented ITC and EPR data consistently demonstrated that HRG has 10-12 metal ion binding sites of equal and high affinity, which are presumably located in its HRR. Interestingly, a mass spectrometry study on a 35 residue anti-angiogenic HRG peptide derived from the HRR concluded that Zn II binding involves various locations within this region, rather than one single preferred site, 69 thus strongly supporting the hypothesis of different binding sites in the HRR being randomly occupied.