5.2.1 Materials
The trans complexes tHpz, tMeim and tMepz were used as prepared in Chapter 3. Cisplatin was obtained from Sigma-Aldrich. The stock solutions of the platinum complexes at a concentration of 1 x 10-4 M in NaClO4 (10 mM) were prepared at 25 ºC. Calf Thymus DNA
(CT DNA) (42% G + C, molecular mass about 20 kDa) was prepared and characterized, as described in literature.8,9 Plasmid pSP73KB (2464 bp) was isolated according to standard procedures and banded twice in CsCl/ethidium bromide (EtBr) equilibrium density gradients.10 Ethidium bromide (EtBr), thiourea, agarose and NaCl were used as obtained from Merck. The radioactive products were obtained from Amersham.
5.2.2 Platination reactions
CT DNA was incubated with the platinum complexes in 10 mM NaClO4 at 37 ºC for
24 hr in the dark. At various time intervals different aliquots of the reaction mixture were taken and precipitated by ethanol and redissolved in 10 mM NaClO4 for a later analysis by
differential pulse polarography (DPP), to determine the amount of platinum that did not react with DNA. An aliquot of these samples was used to determine the value of rb (rb is defined as
the number of molecules of platinum bound per nucleotide residue) for each of the complexes.11
5.2.3 Unwinding of negatively supercoiled DNA
The unwinding of the closed circular supercoiled pSP73 plasmid DNA was studied by an agarose gel mobility shift assay.12 The unwinding angle Φ induced per platinum-DNA adduct was calculated from the determination of the rb(c) value at which the complete
transformation of the supercoiled to relaxed form of the plasmid was attained. Samples of pSP73KB plasmid were incubated with the tHpz, tMeim and tMepz at 37 ºC in the dark for 24 hr at different rb values (0.10-0.16 for complex tHpz and 0.01-0.12 for complexes tMeim
and tMepz). To all samples 1 μl of Tris/acetate/edta buffer (TAE) (0.04 M Tris-acetate, 0.001 M edta, pH 7.0) and 2 μl of gel-loading buffer (0.25 % bromophenol blue, 40 % (w/v sucrose in water)) were added. All samples were subjected to electrophoresis on 1 % agarose gels running at 25 ºC with the TAE buffer; the voltage was set at 20 V. The gels were then stained with EtBr and read by photography.
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5.2.4 DNA interstrand cross-links assay
Complexes tHpz, tMeim and tMepz were incubated, at different rb values, with 0.1
μg of pSP73KB DNA linearized by EcoRI for 24 hr at 37 ºC in the dark. The linear duplexes were first 3’-labeled by means of a Klenow fragment of DNA polymerase I in the presence of [α-32P]dATP. To all the samples 0.6 μl of 1 M NaOH and 1,2 μl bromophenol blue (loading buffer: 33 mM NaCl, 70 mM Tris-HCl, 1 mM edta, 2 % (w/v) SDS, 0.01 % (w/v) bromophenol blue, 10 % glycol, pH 6.8)) were added. The amount of interstrand cross-links was analyzed by electrophoresis under denaturing conditions on alkaline agarose gel (1 %). After the electrophoresis was complete, the intensities of the bands corresponding to single strands of DNA and to interstrand cross-links duplex (ICL) were quantified by the FUJIFILM bioimaging analyser, and the radioactivities associated with bands were quantified with the AIDA image analyser software.
The frequency of interstrand cross-links (ICLs), % ICLs/Pt (number of ICLs per adduct), was calculated as % (ICL/Pt) = XL/(4928rb) (pSP73KB contained 4928 nucleotide
residues). XL is the number of ICLs per molecule of the linearized DNA duplex, which was calculated assuming a Poisson distribution of the ICLs, as XL = -ln A, where A is the fraction of molecules running as a band corresponding to the non-cross-linked DNA.
5.2.5 Thiourea assay, estimation of monofunctional adducts
Calf thymus DNA (double-stranded, or thermally denatured), at a concentration of 0.16 mg/ml, was incubated with tHpz, tMeim and tMepz at ri value of 0.1 (ri is defined as the
molar ratio of free metal complex to nucleotide-phosphates at the onset of the incubation). Different aliquots were withdrawn at 10 min, 1, 2, 4, 8, 24 and 48 hr time intervals. Of these
solutions, 40 μl was added to 40 μl of 20 mM thiourea, and another 40 μl, as a control, was added to 40 μl of 1 M NaCl. After 10 min of incubation at 37
ºC, 420 μl of 0.5 M NaCl pH 7.0 was added to both aliquots to stop the reaction. The samples were stored at 4 ºC until all the samples were incubated. Then the samples were exhaustively dialyzed, first twice against 0.5 M NaCl for 1.5 hr and subsequently twice against 10 mM NaClO4 (1.5 hr and overnight). After that, the concentration of DNA was determined by UV
spectroscopy and the concentration of platinum by FAAS.
5.2.6 Fluorescence measurements
Fluorescence measurements were performed on a Shimadzu RF 40 spectrofluorophotometer using 1-cm quartz cells. Fluorescence measurements of DNA modified by platinum in the presence of EtBr were performed at an excitation wavelength of
DNA-binding studies of asymmetric trans-platinum(II) complexes
546 nm, and the emitted fluorescence was analyzed at 590 nm. The fluorescence intensity was measured at 25 ºC in 0.4 M NaCl to avoid secondary binding of EtBr to DNA.13 The used concentrations were 0.04 mg/ml for DNA and 0.04 mg/ml for EtBr, which correspond to the saturation of all intercalation sites of EtBr in DNA.13 The fluorescence intensity was measured after equilibrium for 60 min at 25 ºC in the dark. Full details of this measurement can be found in the original literature.13
5.2.7 Other physical measurements
Absorption spectra were measured with a Beckmann DU-7400 spectrophotometer. FAAS measurements were carried out on a Unicam 939 AA spectrometer with a graphite furnace. DPP curves were recorded with the aid of an EG&C PARC Electrochemical Analyzer, Model 384B. Circular dichroism (CD) spectra were recorded at 25 ºC using a JASCO spectropolarimeter, Model J720. Fluorescence was measured using a Perkin Elmer Luminescence Spectrometer LS50B. The gels were dried and visualized by using the FUJIFILM bioimaging analyzer, and the radioactivities associated with bands were quantified with the AIDA image analyzer software.