A Mass Spectral Study of the Binding of the Anticancer Drug Cisplatin to Ubiquitin

Cisplatin is a widely used antitumor agent for the treatment of testicular and ovarian cancers. It is believed to exert its cytotoxic effect by reacting with DNA. Within one day of injection, 65–98% of the platinum in the blood plasma is protein-bound. Pt–protein adducts are believed to be the cause of the drug's side effects but, to date, nuclear magnetic resonance (NMR), ultraviolet (UV) and Fourier transform infrared (FT-IR) studies of Pt–protein adducts have provided only low resolution data. In the investigation reported herein, ubiquitin was chosen as a model protein to evaluate the utility of electrospray ionization mass spectrometry (ESI-MS) for study of the interactions of Pt anticancer drugs with proteins. Reacting ubiquitin with a 10-fold excess of cisplatin at 37°C resulted in formation of 1:1 adducts after less than 24 h, appearance of nearly equal amounts of 1:1 and 1:2 adducts after six days and persistence of 1:1 adducts even after 15 days. The Pt binding sites are probably Met1 and His68. Four monoadducts were characterized; Pt(NH₃)₂(Cl)(Ub)M_r 8829, Pt(NH₃)₂(H₂O)(Ub)M_r 8812, Pt(NH₃)₂(Ub)M_r 8892, Pt(NH₃)(Ub)M_r 8875. In the first two adducts, platinum is bound to ubiquitin in a monodentate fashion, the third in a bidentate fashion and the fourth in a tridentate (internally crosslinked) manner. Different Pt complexes form different adducts. Cisplatin forms four monoadducts and several diadducts. Transplatin forms mainly one monodentate monoadduct [trans-Pt(NH₃)₂ (Cl)(Ub)], while Pt(en)Cl₂ forms mainly a bidentate monoadduct [Pt(en)Ub)]. Platination affects the protein structure. Formation of the bidentate adduct (M_r 8792) does not unfold the protein, whereas generation of the tridentate adduct (M_r 8775) unfolds the protein and gives rise to two charge state distributions. This can be due to having two conformations for the same cross-link or having two different cross-links. Based on the results of this mass spectral study, a possible mechanism for the binding of cisplatin to ubiquitin is proposed.