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Eur. J. Mass Spectrom. 5, 501 - 510 (1999) |
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| A mass spectral study of the binding of the anticancer drug cisplatin to ubiquitin | ||
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Dan Gibson and Catherine E. Costello |
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| ABSTRACT: | ||
| 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 37ụC 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) Mr 8829, Pt(NH3)2(H2O)(Ub) Mr 8812, Pt(NH3)2(Ub) Mr 8892, Pt(NH3)(Ub) Mr 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 (Mr 8792) does not unfold the protein, whereas generation of the tridentate adduct (Mr 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. | ||
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Keywords: cisplatin, platinum-based anticancer agents, protein binding, metal-protein adducts, ESI-MS, mechanisms of interaction |
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