EXPLORING THE INTERACTIONS BETWEEN PHOSPHANE-BASED
PLATINUM(II) DICHLORIDES AND CYTOCHROME C BY ESI-HRMS
Michelucci E.
1, Mügge C.
2, Gabbiani C.
3, Boscaro F.
1, Messori L.
3, Weigand W.
21 Mass Spectrometry Center (CISM), University of Florence, Via U. Schiff 6, 50019 Sesto Fiorentino, Firenze (Italy).
2 Institute of Inorganic and Analytical Chemistry, Friedrich-Schiller-University Jena, August-Bebel-Strasse 2, 07743 Jena (Germany).
3 Laboratory of Metals in Medicine, Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze (Italy).
INTRODUCTION
Electrospray ionization high resolution mass spectrometry (ESI-HRMS) represents today a very powerful technique to analyse metallodrug/protein interactions and to elucidate the nature of the resulting adducts at the molecular level.
1 2
Fig. 1. cis-(Me3P)2PtCl2 (1) and cis-(Et3P)2PtCl2 (2).
REFERENCES
1. Casini A. et al. Biochemistry, 2007, 46, 12220-12230.
2. Zhao T., King F.L. J. Am. Soc. Mass Spectrom., 2009, 20, 1141-1147.
CONCLUSIONS
ESI-HRMS proved to be an useful instrument to characterise diphosphane platinum(II) dichlorides 1- and 2-cyt c adducts and interactions. A number of conclusions may be drawn from the analysis of the above results:
the phosphane ligand is invariably kept while both chloride ligands are released upon adduct formation;
platinum phosphanes 1 and 2 manifest a very high efficiency in cyt c metalation mainly giving rise to stable 3:1 Pt-protein adducts (three primary binding sites on cyt c) at variance with classical anticancer platinum drugs which mainly form monoadducts2;
cyt c + 3[Pt(PEt3)2] adduct enzymatic digestion experiments clearly show that the three platinum fragments are contained in G1-T49 portion of cyt c and support the idea that histidines H18, H26 and H33 are probably the preferential binding sites for 2 on cyt c. Apparently, the presence of the phosphane ligands confers to platinum(II) center a pronounced selectivity for histidine coordination on cyt c at variance with classical anticancer platinum drugs that preferentially bind to M652.
INSTRUMENTAL CONDITIONS
ESI-HRMS spectra were acquired using a LTQ-Orbitrap high-resolution mass spectrometer equipped with a conventional ESI source. Working conditions: direct introduction of the samples (flow rate 5 μL/min), spray voltage 3.1 kV, capillary voltage 45 V, capillary temperature 220 °C, sheath gas 17 a.u., auxiliary gas 1 a.u., nominal resolution 100000 at m/z 400.
While exploring the interactions between the model protein cytochrome c (cyt c) and new phosphane-based platinum(II) complexes for cancer therapy, we found out a remarkable reactivity for two diphosphane platinum(II) dichlorides, namely cis-bis(trimethylphosphane) platinum(II) dichloride (1) and cis-bis(triethylphosphane) platinum(II) dichloride (2) (fig. 1). A deeper investigation on the nature of the adducts formed by 1 and 2 and the determination of their binding sites on cyt c (fig. 2) are the object of this study.
RESULTS AND DISCUSSION
Cyt c interaction studies
Samples were prepared by dissolving cyt c (100 µM) in 25 mM tetramethylammonium acetate buffer (pH 7.4). The selected complex was added to this solution to metal:protein ratios of 1:1, 2:1, 3:1 and 5:1. The samples were incubated at 37 °C for 72 h and diluted 20-fold with water before MS analysis.
Selected deconvoluted spectra (fig. 3) reveal that phosphane ligands are kept while both chloride ligands are released upon adduct formation. Even though lower adduct stoichiometries are evident in the 1:1 incubated samples, a clear trend is visible towards eventually forming 3:1 adducts for both complexes. Higher stoichiometry adducts are almost void. Therefore, a specific binding mechanism and three defined binding sites on cyt c must exist for 1 and 2.
Digestion experiments
In order to determine the nature of binding sites, digestion experiments were carried out on cyt c reacted with complex 2 in 1:3 ratio (solution b, fig. 3) and on cyt c (control) using Endoproteinase Asp-N1 and trypsin2.
Endoproteinase Asp-N digestion experiment (fig. 4) clearly show that cyt c segment G1-T49 is nearly completely reacted with three equivalents of Pt(PEt3)2 whereas the second segment D50-E104 shows no platinum adducts. A comparison between experimental data from tryptic digestion experiment (fig. 5) and theoretical simulations (data not shown) confirms the presence of two Pt(PEt3)2 moieties on G23-R38 fragment of cyt c (containing H26 and H33) and the presence of one Pt(PEt3)2 unit on the fragment C14-K22 (containing H18, simply coordinated and not covalently bound to heme group).
1 10 20 30 40 50 60 70 80 90 100 104
GDVEKGKKIF VQKCAQCHTV EKGGKHKTGP NLHGLFGRKT GQAPGFTYTD ANKNKGITWK EETLMEYLEN PKKYIPGTKM IFAGIKKKTE REDLIAYLKK ATNE
Fig. 2. Horse heart cyt c aminoacidic sequence. In red, blue and green are highlighted the residues that manifest affinity for Pt(II) compounds, namely Cys, His, Met. In red (C14
and C17) and in blue (H18 and M80) the residues covalently bound with and coordinated to heme group in horse heart cyt c, respectively. In green (H26, H33, M65) the remaining potential reaction sites to form Pt(II) adducts.1
Fig. 3. Deconvoluted ESI-HRMS spectra of 2 incubated for
72h at a 1:1 (a), 3:1 (b), 5:1 (c) metal: protein ratio.
12400 12800 13200 13600 14000 14400 Da 0 50 100 0 50 100 R el at iv e A bu nd an ce 0 50 100 12358.33 Cyt c 13646.71 Cyt c + 3Pt(PEt3)2 13217.58 Cyt c + 2Pt(PEt3)2 12788.46 Cyt c + Pt(PEt3)2 13646.72 Cyt c + 3Pt(PEt3)2 13216.59 Cyt c + 2Pt(PEt3)2 14074.85 12787.45 14542.95 12358.33 13646.73 Cyt c + 3Pt(PEt3)2 14541.97 Cyt c + 4Pt(PEt3)2 + Pt(PEt3)2Cl 14075.86 Cyt c + 4Pt(PEt3)2 13217.60 Cyt c + 2Pt(PEt3)2 12787.46 12358.33 a 1:1 ratio b 3:1 ratio c 5:1 ratio
Fig. 4. Endoproteinase Asp-N digestion: deconvoluted
ESI-HRMS spectra of free cyt c (top) and solution b of fig. 3 (bottom).
m/z R el at iv e A bu nd an ce 1040 1080 1120 0 1160 1200 1240 1280 4 12 8 16 20 0 4 8 12 16 20 1168.63 z=1 T28-R38 1168.62 z=1 T28-R38 1041.52 z=2 G56-K72 1041.52 z=2 G56-K72 1092.63 z=1 E92-K100 1092.63 z=1 E92-K100 1105.57 z=2 G56-K73 1105.57 z=2 G56-K73 1113.52 z=1 1130.36 z=1 1151.58 z=1 1158.35 z=1 1184.62z=1 1200.61 z=1 1113.52 z=1 1151.58 z=1 1190.60 z=1 1231.40 z=1 1259.40 z=1 1264.66 z=1 1032.38 z=2 C14-K22 + heme + Pt(PEt3)2 1053.53z=2 1060.50 z=2 1124.55 z=2 1184.56 z=1 1219.64 z=1 1246.95 z=2 1255.95 z=2 1268.10 z=2 G23-R38 + 2Pt(PEt3)2
Fig. 5. Tryptic digestion: enlargements of ESI-HRMS
spectra. Top: free cyt c; bottom: solution b of fig. 3.
6000 6400 6800 7200 Da 0 50 1000 50 100 R el at iv e A bu nd an ce 5942.92 G1-T49 6372.05 G1-T49 + Pt(PEt3)2 6432.41 D50-E104 7231.31 G1-T49 + 3Pt(PEt3)2 6801.18 G1-T49 + 2Pt(PEt3)2 5942.97 G1-T49 6432.47 D50-E104
