Direct determination of the primary binding site of cisplatin on cytochrome C by mass spectrometry.
Protein-cisplatin interactions lie at the heart of both the effectiveness of cisplatin as a therapeutic agent and side effects associated with cisplatin treatment. Because a greater understanding of the protein-cisplatin interactions at the molecular level can inform the design of cisplatin-like agents for future use, mass spectrometric determination of the binding site of cisplatin on a model protein, cytochrome c, was undertaken in this paper. The monoadduct cytochrome c-Pt(NH(3))(2)(H(2)O) is found to be the primary adduct produced by the cytochrome c-cisplatin interactions under native conditions. To locate the primary binding site of cisplatin, both free cytochrome c and the cytochrome c adducts underwent trypsin digestion, followed by Fourier transform mass spectrometry (FT-MS) to identify unique fragments in the adduct digest. Four such fragments were found in the adduct digest. Tandem mass spectrometry (MS/MS and MS(3) indicates that two fragments are Pt(NH(3))(2)(H(2)O) bound peptides (Gly56-Glu104 and Asn54-Glu104) with one water associated at the peptide bond Lys79-Met80, and the other two fragments are heme containing peptides (acety1-Gly1-Lys53 and acety1-Gly1-Lys55). The product-ion spectra of the four fragments reveal that Met65 is the primary binding site of cisplatin on cytochrome c.
Zhao
Ting
T
C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, West Virginia 26506-6045, USA.
King
Fred L
FL
D000818:Animals; D001665:Binding Sites; D002945:Cisplatin / Q000737:chemistry* / Q000378:metabolism; D045304:Cytochromes c / Q000737:chemistry* / Q000378:metabolism; D005583:Fourier Analysis; D006736:Horses; D013058:Mass Spectrometry / Q000379:methods*; D008715:Methionine / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism
D045304:Cytochromes c; D008715:Methionine; D014357:Trypsin; D002945:Cisplatin