DLS and Urea Gel Analysis of Protein Complexes

Changes in size of proteins following the reactions of apoTf and Fe₂Tf with Ru complexes were determined by a dynamic light scattering (DLS) technique, using a Malvern ZetaSizer NanoS instrument (173° scattering angle, 298 K) with ZEN0040 disposable cuvettes (Malvern Panalytical, Malvern, UK). Protein samples (0.10 mM) were diluted 10-fold with binding buffer before the measurements. The measured parameters were the averages of 12–15 scans (scan time, 3 s). Urea gel electrophoresis of apoTf and Fe₂Tf samples [28 (link)] in the presence of varying concentrations of KP1019 was performed as described previously [29 (link)]. Analysis of Tf crystal structures that are available in the public domain from the Protein Data Bank (PDB) was performed with PyMOL software (version 2.1.1, Schrodinger LLC 2021, New York, NY, USA).