Zinc Binding Affinity of SLC39A7 Peptides

SLC39A7 peptides that contain either no (Me0; HGHSHAHGHGHTH) or six 1MH (Me6, HGXSXAXGXGXTX; X = 1MH)) residues were synthesized with a peptide synthesizer (ABI, 433 A) using Fmoc-3-methyl-L-histidine (Merck) for the 1MH residue. The peptides were purified with HPLC, and purity determined by MALDI-MS. Dry weights were quantified with an amino acid analyzer (Hitachi, L-8900) for the calculation of molarity. ITC assay was performed as reported previously^{51 (link)}, with a slight modification. Briefly, SLC39A7 peptides and Zn²⁺ solutions were prepared in 20 mM Tris –HCl pH 7.5, 100 mM NaCl. Titration of ZnCl₂ (2 mM) into peptide solutions (50 μM) was performed at 30 °C using an ITC titration calorimeter (Malvern Panalytical, MicroCal iTC200). 39 injections of 1 µL were made with an equilibration time of 1 min between injections. Integration of the thermograms after correction for heats of dilution yielded binding isotherms that fit best to a one-set of sites binding model using the ITC data analysis software Origin 7.0 (MicroCal Inc., Piscataway, NJ). A non-linear least-squares curve-fitting algorithm was used to determine the stoichiometric ratio (n), the dissociation constant (K_d), and the change in enthalpy (ΔH) of the interaction. All ITC experiments were performed in triplicate.