EPR spectroscopy requires the presence of a paramagnetic species in the sample. Binding of diamagnetic Zn II can therefore not be examined by EPR directly, however previous studies of HRG or HRG-derived peptides have reportedly used the paramagnetic Cu II instead. 17, To confirm the validity of this approach, we demonstrated that ITC yields very similar numbers of binding sites and binding affinities in vitro when comparing paramagnetic Cu II (this study, Figure ) and diamagnetic (EPR silent) Zn II . Next, a heparin binding assay confirmed a similar increase in the affinity of HRG binding to heparin for both, Zn II and Cu II , indicating the exertion of a similar functional effect (Figure ).