Interactions of anti-cancer Ru(III)-complexes, KP1019, KP418, and NAMI-A, with human serum transferrin (hsTf) and albumin (hsA), and their speciation in serum were characterized. NAMI-A was distinguished from KP1019 and KP418 by the instability of its Ru(III) oxidation state. EPR studies suggested a ligand-exchange mediated binding of KP1019 and KP418 to hsTf and hsA. With hsA a hydrophobic interaction was also detected. KP1019 exhibited a different speciation profile in serum from that of KP418. UV-visible studies demonstrated a faster hsTf-binding ability of KP1019 over KP418. It was proposed that these differences might partially explain the disparate anti-cancer activities of KP1019 and KP418. EPR analysis of KP1019-binding to the His249Ala mutant of hsTf and to diferric-hsTf revealed a Ru(III)-state stabilizing role of the iron-binding site of hsTf in KP1019 binding. In addition, a procedure for expression and purification of full-length recombinant hsTf in P. pastoris was devised
