Determination of binding sites in carboplatin-bound cytochrome c using electrospray ionization mass spectrometry and tandem mass spectrometry.
Interaction of carboplatin with cytochrome c (Cyt. c) has been investigated by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS). ESI-MS studies revealed that the ring-opened adducts of carboplatin with Cyt. c were formed in the stoichiometric ratio of 1:1 and 2:1 at pH 5.0 and 37 degrees C and in the stoichiometric ratio of 1:1 only at pH 7.0 and 37 degrees C. It was also found that Cyt. c could be cleaved by carboplatin at pH 2.5 and 50 degrees C. The cleaved fragments of Cyt. c were determined by ESI-MS and MS/MS analysis to be Glu66 approximately Met80, Ac-Gly01 approximately Met65, Glu66 approximately Glu104, Ac-Gly01 approximately Met80 and Ile81 approximately Glu104. The carboplatin prefers to anchor to Met65 first, then to Met80. To further confirm the binding site of Met, AcMet-Gly was used as the model molecule to investigate its interaction with carboplatin and its hydrolysis reaction. On the basis of species detected during the reaction monitored by ESI-MS, a possible pathway of the cleavage reaction was proposed.
Yang
Gaosheng
G
State Key Laboratory of Coordination Chemistry, Coordination Chemistry Institute, Nanjing University, Nanjing 210 093, China.
Miao
Ren
R
Jin
Chen
C
Mei
Yuhua
Y
Tang
Huiwei
H
Hong
Jin
J
Guo
Zijian
Z
Zhu
Longgen
L
D000595:Amino Acid Sequence; D001665:Binding Sites; D016190:Carboplatin / Q000737:chemistry* / Q000378:metabolism*; D045304:Cytochromes c / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization
D045304:Cytochromes c; D016190:Carboplatin