Journal of Inorganic Biochemistry 91 (2002) 306–311
www.elsevier.com/ locate / jinorgbio
I nteractions of cisplatin and transplatin with proteins.
Comparison of binding kinetics, binding sites and reactivity of the Pt-
protein adducts of cisplatin and transplatin towards biological nucleophiles
,1*Tal Peleg-Shulman, Yousef Najajreh, Dan Gibson
Department of Medicinal Chemistry and Natural Products, School of Pharmacy, PO Box 12065, The Hebrew University of Jerusalem,
Jerusalem 91120, Israel
Received 27 September 2001; received in revised form 26 November 2001; accepted 18 December 2001
Abstract
In this manuscript we report on the interactions of cis-DDP (cisplatin, cis-diamminedichloroplatinum(II)) and trans-DDP (transplatin,
trans-diamminedichloroplatinum(II)) with two model proteins, ubiquitin (Ub) and horse heart myoglobin (Mb), and attempt to answer the
question whether proteins that have methionine-Pt adducts can transfer the platinum to biological nucleophiles and particularly to DNA.
Our study shows that cisplatin and transplatin form different adducts with ubiquitin: transplatin forms one major adduct, trans-
[Pt(Ub)(NH ) Cl], while cisplatin forms four distinct adducts, [Pt(Ub)(NH ) Cl], [Pt(Ub)(NH ) (H O)], [Pt(Ub)(NH ) ], and3 2 3 2 3 2 2 3 2
[Pt(Ub)(NH )]. When binding ubiquitin, Met1 is the preferred binding site of cisplatin, but not of transplatin. Cisplatin binds faster than3
transplatin to both ubiquitin and horse heart myoglobin. Both cisplatin and transplatin adducts form stable ternary adducts when reacted
with 59-guanosine monophosphate (59-GMP) or a tetranucleotide. No transfer of the Pt moiety from the proteins to the nucleotides was
observed. Glutathione efficiently removes the platinum from preformed adducts of both cisplatin and transplatin with ubiquitin.  2002
Elsevier Science Inc. All rights reserved.
Keywords: Cisplatin; Transplatin; Protein binding; Electrospray ionization mass spectrometry (ESI-MS)
1 . Introduction [1]. Cisplatin is believed to induce apoptosis in cancer cells
by covalently modifying the DNA [2]. Its geometric
Cisplatin (Fig. 1a) is a widely used anti-tumor agent that isomer, trans-[Pt(NH ) Cl ] (Fig. 1b), has no cytotoxic3 2 2
is used in the clinic to treat testicular and ovarian cancers activity [3]. The difference in antitumor activity between
the two isomers is attributed to the inability of the trans
isomer to form 1,2-GpG intrastrand crosslinks due the 1808
angle between its two semi-labile chloride ligands. Cellular
proteins such as the HMG box proteins recognize cisplatin
modified DNA and bind to it. The binding of the proteins
to the platinated DNA can serve to protect the DNA from
being repaired by nuclear excision repair enzymes, or
alternatively, hijack the proteins from their normal func-
tion [4,5]. Transplatin modified DNA is not recognized by
those same cellular proteins [6,7].
Cisplatin is administered intravenously, and within 1
Fig. 1. (a) cisplatin; (b) transplatin. day, 65–98% of the drug is bound to blood plasma
proteins [8]. While Pt-DNA adducts are believed to be
responsible for the drug’s cytotoxicity, the exact role that
*Corresponding author. Tel.: 1972-2-675-8702; fax: 1972-2-675- Pt-protein adducts play in the mechanism of action of the
7076. drug is yet to be elucidated. It has been postulated that inE-mail address: gibson@md2.huji.ac.il (D. Gibson).
1 addition to drug inactivation, cisplatin binding to proteinsAffiliated with the David R. Bloom Center for Pharmacy at The
Hebrew University of Jerusalem, Jerusalem, Israel. may be the cause of many of the drug’s side-effects [9,10].
0162-0134/02/$ – see front matter  2002 Elsevier Science Inc. All rights reserved.
PI I : S0162-0134( 02 )00362-8

T. Peleg-Shulman et al. / Journal of Inorganic Biochemistry 91 (2002) 306 –311 307
Other reports suggest that Pt-HSA (human serum albumin) cleophiles such as glutathione and 59-guanosine mono-
adducts may be important for the activity of the drug [11]. phosphate are reported.
When preformed Pt-HSA adducts were administered clini-
cally they increased the survival time of the patients. Also,
2 . Experimentalpatients with low levels of HSA did not respond well to
cisplatin based chemotherapy [12]. It is clear that the
2 .1. Materialsformation of Pt-protein adducts, that effectively competes
with formation of the cytotoxic Pt-DNA lesions, can
Cis- and trans-DDP, ubiquitin and horse heart myoglo-reduce the efficacy of Pt antitumor agents.
bin were all purchased from Sigma–Aldrich (Israel) andThe efficient binding of platinum complexes to proteins
were used without further purification.and peptides is not surprising since platinum has a high
affinity for sulfur containing ligands such as methionine
2 .2. Platination reactionsand cysteine [13]. The abundance of extra- and intra-
cellular platinophiles makes it difficult to understand how
Platination reactions were carried out at 1–2 mMPt even reaches the DNA. Model studies suggest that a
concentrations, in 10 mM phosphate buffer, pH 6.4, 37 8C.platinum moiety that is bound to a methionine thioether
Excess platinum was removed by ultra-filtration usingcan be transferred to the N7 of the guanine of single
Microcon YM-3 centrifugal filter devices at 4 8C andstranded and double stranded oligonucleotides (but not
12,000 rpm, prior to all NMR and adduct-reactivityfrom the cysteine thiolate) [14–17]. On the basis of these
studies. Kinetics by ESI-MS were measured directly on thestudies it was suggested that proteins that form Pt-
reaction mixtures following ZipTip
(C , Millipore)18methionine adducts could act as a platinum reservoir for
treatment.subsequent DNA platination [13]. Many studies on the
reaction of platinum complexes with amino acids and 2 .3. Reactions of Pt-protein adducts with nucleophilesproteins have been reported [18–20], but there are few
high-resolution reports on the interactions of cis-DDP A 2 mM concentration of Ub-Pt adducts in 10 mM(cisplatin, cis-diamminedichloroplatinum(II)) or trans- phosphate buffer, pH 6.4, free of any unreacted platinumDDP (transplatin, trans-diamminedichloroplatinum(II)) (removed by ultrafiltration), was reacted with a five-fold
with proteins.
excess of the relevant biological nucleophile or oligo-We have recently shown that electrospray ionization
nucleotide. The reactions were at 37 8C. Kinetics by ESI-
mass spectrometry is an extremely useful technique for MS were measured directly on the reaction mixtures
studying the interactions of cisplatin with proteins [21], following ZipTip
(C , Millipore) treatment.18and provides information on the nature of the Pt-protein
1 15
adducts that are formed. [ H, N] heteronuclear single 2 .4. ESI-MS
quantum coherence (HSQC) NMR spectroscopy has been
15
utilized in the analysis of N-labeled platinum ammine Electrospray ionization mass spectrometry was mea-
complexes providing information on the nature of the sured on a ThermoQuest Finnigan LCQ-Duo in the15ligands that are trans to the N-labeled ammine [22]. The positive ion mode. Elution was in 49:49:2 water:
combination of the two techniques provides a powerful methanol:acetic acid at a flow rate of 15 ml /min. Samples
tool for simultaneously studying the modifications of the of the platination reactions and adduct reactivity studies
protein (electrospray ionization mass spectrometry, ESI- were diluted 100-fold prior to ESI-MS analysis. Data were
MS) and the changes in the platinum coordination sphere processed using ThermoQuest Finnigan’s Xcalibur
(HSQC) [23]. Biomass Calculation and Deconvolution software.
Since Pt-protein adducts are important in defining the
therapeutic profiles of the drugs, it is important to under- 2 .5. Oxidation of ubiquitin
stand the basic principles that govern the formation and
reactivity of these adducts. In this manuscript we report on Performic acid oxidation of the Met1 residue of
the interaction of cis- and trans-DDP with model proteins, ubiquitin was performed according to the method of
ubiquitin (Ub) and horse heart myoglobin (Mb). These Breslow et al. [24].
proteins were chosen because both are well characterized,
have methionines and histidines (but not cysteines) and are
good candidates to try and answer the question whether 3 . Results and discussion
proteins that have methionine-Pt adducts can transfer the
Pt to biological nucleophiles and particularly to DNA. The 3 .1. The types of adducts formed by cis- and trans-DDP
binding kinetics, the nature of the adducts formed, the with ubiquitin
binding sites of the complexes and the reactivity of the
Pt-protein adducts towards biologically relevant nu- Electrospray ionization mass spectrometry is a soft