CD and DSC Investigation of Individual and Complex Influence of Meso-Tetra(4-Oxiethylpyridil) Porphyrin (TOEPyP4) and Its Zn-Complex on DNA

Jamlet Monaselidze, Evgeni Kiziria, Maya Gorgoshidze, David Khachidze, Maya Kiladze, Eteri Lomidze, Hakob Margaryan, Nune Hakobyan

Department of Radiophysics, Erevan State University, Erevan, Armenia.
E. Andronikashvili Institute of Physics, Tbilisi State University, Tbilisi, Georgia.

DOI: 10.4236/ajac.2012.310092                

Abstract

CD spectra of (DNA-TOEPyP4) + ZnTOEPyP4, (DNA-ZnTOEPyP4) + TOEPyP4, and DNA + (TOEPyP4-ZnTO- EPyP4) complexes have been studied. It is shown that CD spectra of these triple complexes significantly differ from the DNA-TOEPyP4 and DNA-ZnTOEPyP4 double complex spectra, and they are not sum of these double complexes. Especially some strong differences in CD spectra of the triple and double complexes were observed when both porphyrins were added simultaneously into the DNA solution. In this case, ZnTOEPyP4 revealed a dominant influence on CD spectrum form. Zn-porphyrin also caused a strong intensity of positive band at 416 nm and a negative band at 437 nm when it was added into solution containing the DNA-TOEPyP4 complex. On the basis of obtained data, it was supposed that the observed significant changes in CD spectra of triple complexes were connected to an altered DNA conformation initiated by intercalation of porphyrin TOEPyP4 into GC-rich sites. The melting process analysis of the double complexes was carried out. The mechanisms of individual and joint influence of the porphyrins on DNA, and influence of binding modes on stability of these complexes are also discussed.

Keywords

CD; Absorption; Microcalorimetry; Porphyrin

1. Introduction

Among ligands, a special attention is paid to the water soluble cation meso-tetra(4-N-oxyethylpyridil) porphyrins-TMPyP4 type porphyrins-because it is minimal toxic. It predominantly accumulates in tumor cells and binds to G-quadruplex of telomeric DNA [1,2], and defends it from high telomerase activity detaining growth of transformed cells in case of many leukemic diseases [3,4]. Its metal complexes can induce DNA strand cleavage at deoxyribose residues and initiate apoptosis of cancer cells [5]. In the classic works [6-9], the absorption mechanisms of the TMPyP4 porphyrins into DNA, AT and GT oligomers, and polynucleotide were investigated with the help of different physical technologies. The following have been demonstrated: intercalation, outside self-stacking and outside random binding of porphyrins to DNA. It was shown [7] that porphyrin plane molecules TMPyP intercalate between the base pairs of DNA double helix and they bind only to 5’CG3’ areas, and not to other sequences. Outside binding modes were studied in works [6-11]. It was also shown that these binding modes were formed mainly by porphyrins having axial ligands, which were absorbed on AT rich areas located in minor and major grooves, as well as double helix surface of DNA [12]. The outside random binding of TMPyP to DNA happens due to interaction of the positive charge of porphyrin pyrydil ring with the negative charge of phosphate groups. The self-stacking was studied in series of works [9,13]. It was shown that just this binding mode was the main reason for DNA aggregation. This is also connected to the electrostatic interaction between porphyrin and phosphate groups, but the strong intraplane interaction between porphyrins and DNA have the main contribution in formation of this mode.

The obtained results gave us a possibility to deeper understanding of TMPyP binding mechanism, including TMPyP binding to DNA in vitro and in situ, which is very important for understanding influence on chromatin DNA in live cells. In the given work we tried to characterize the joint influence of two different conformational conditions of TOEPyP4-plane porphyrin and its nonplane Zn-complex. TOEPyP4 is an analogue of TMPyP, according to its physical and chemical properties [14,15].

These investigations are important from medical point of view, because in case of many diseases, DNA in vivo may be attacked simultaneously not only by endogenes and exogenes porphyrins free from metals, but also by metal-containing porphyrins, some small proteins, as well as transition metal ions that can change the binding mode of porphyrins to DNA [3,16,17]. This can change the therapeutic effect of the drug, especially in case of photodynamic therapy. Therefore, we think that it is very important to know the mechanisms of combined influence of intercalating and non-intercalating (outside binding) porphyrins on DNA.

2. Materials and Methods

The water-soluble meso-tetra(4-N-oxyethylpyridil) porpyrin (TOEPyP4) and its Zn-containing analogue was synthesized in ESU according to the method described in work [18], and it was kindly granted by professor S. Haroutiunian. Calf thymus DNA was purchased from Sigma. Porphyrin concentration was determined spectrophotometrically by following coefficients of TOEPyP4: M_w = 940 Da, ε₄₂₂ = 2.1 × 10⁵ M^–1·cm^–1; and ZnTOEPyP4: M_w = 1003 Da, ε₄₄₀ = 1.3 × 10⁵ M^–1·cm^–1, where M_w is molecular weight, and ε₄₂₂ and ε₄₄₀ are Sore absorption bands initiated by binding of porphyrin to DNA. All studies were carried out in buffer solution 10 mM NaCl, 1 mM Na-phosphate, and Ph 7.02. DNA concentration was determined spectrophotometrically using the molar extinction coefficient ε₂₅₈ = 6700 cm^–1·M^–1. The mixed ratio-r-was defined as molar ratio of porphyrin to DNA base pair. Absorption spectra were recorded on spectrophotometer UNICAM SP 1800 (UK) using 1.0 cm quartz cells. CD spectra were recorded on spectropolarimeter JASCO 500A at 20˚C using 1.0 cm quartz cells (CD is shown in millidegree of ellipticity). DNA concentration was 0.30 µM in all CD experiments. The doublehelix DNA-porphyrin complex heat properties were measured on DSC. The sensitivity was 0.1 µW, cell volume was 0.2 cm³, scanning rate was 0.75˚C/min, measuring interval was 20˚C - 140˚C [19,20].

3. Results

3.1. Absorption Spectra

Figure 1 presents an absorption spectra of TOEPyP4 and DNA-TOEPyP4 complex at r = 0.052. DNA concentration was 50.1 µM. As it is seen from the figure, addition of porphyrin leads to a significant batochrome shift, which is connected with intercalation of TOEPyP4 into GC rich DNA sites [6,21].

Conflicts of Interest

The authors declare no conflicts of interest.

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