Zinc tetraruthenated porphyrin binding and photoinduced oxidation of calf-thymus DNA

doi:10.1016/S0162-0134(00)00053-2

Journal of Inorganic Biochemistry

Volume 78, Issue 4, 31 March 2000, Pages 269-273

https://doi.org/10.1016/S0162-0134(00)00053-2 Get rights and content

Abstract

The photooxidation of calf-thymus DNA has been investigated in the presence of a supramolecular tetraruthenated zincporphyrin (ZnTRP) sensitizer. A strong interaction of ZnTRP with DNA has been observed, exhibiting a gradual transition from a non-specific electrostatic binding mode to a more specific one at high DNA concentrations. Formation of O₂(¹Δ_g) has been detected from its near-infrared emission, after the excitation of ZnTRP in dioxygen-containing solutions. In the presence of DNA and dioxygen, ZnTRP promotes efficient photocatalytic oxidation of the 2′-deoxyguanosine sites, via their direct reaction with O₂(¹Δ_g), as in a previous work on the ZnTRP-photoinduced oxidation of the free nucleosides.

Introduction

Recent work from our laboratories has focused on the plasmid pBR322 and DNA interactions with a free-base tetraruthenated porphyrin species (TRP), revealing the occurrence of light-induced single-strand breaks and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) formation in the presence of molecular oxygen [1]. The special sensitizer employed combines the remarkable redox and photochemical/photophysical properties of the porphyrins and ruthenium(II)–polypyridine compounds, in addition to their ability to bind DNA via intercalation and outside association [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19]. From the supramolecular point of view, the peripheral ruthenium–polypyridine groups interact with the porphyrin center, promoting energy and electron transfer processes, and enhancing the photodynamic properties of the molecule [20].

Two types of mechanisms have been associated with the photosensitized reactions involving DNA [21]. The so-called Type I mechanism involves direct electron transfer or hydrogen abstraction of the 2′-deoxyguanosine (dGuo) bases by the excited sensitizer, while Type II proceeds via their reaction with singlet oxygen, O₂(¹Δ_g), yielding 8-oxodGuo and 4-hydroxy-8-oxo-4,8-dihydro-2′-deoxyguanosine. Recently, the photodynamic properties of TRP and its zinc-metallated derivative, ZnTRP (Fig. 1), have been evaluated in comparison with methylene blue and riboflavin, using 2′-deoxyguanosine as a model compound [22], [23]. Riboflavin is a typical Type I photosensitizer, while methylene blue exhibits a Type II behavior. The selectivity measured by the ratio of the amount of photoproducts generated by Type II/Type I mechanisms was 0.4 for riboflavin, and 2.3, 3.6 and 5.6 for TRP, methylene blue and ZnTRP, respectively, showing that ZnTRP is the most specific Type II photosensitizer of the series. This interesting result obtained with the dGuo model compound has prompted the present investigation on the molecular interaction and photodynamic properties of the ZnTRP–DNA system.

Section snippets

Chemical

ZnTRP was obtained as previously reported [24]. 2′-Deoxyguanosine was purchased from Pharma-Waldorf (Geneva, Switzerland); calf-thymus DNA (CT-DNA), nuclease P1, alkaline phosphatase (Sigma, Missouri, St Louis) and all other reagents were analytical grade and used as supplied. All solutions were prepared using bidistilled deionized MilliQ (Millipore, Milford, MA) water and kept in the dark at 4 °C.

Fluorescence and electronic absorption measurements

Electronic absorption and luminescence spectra were recorded on a Hewlett-Packard model 8453A

Interaction of ZnTRP with DNA

The binding of ZnTRP to CT-DNA was monitored from the corresponding DNA-induced changes on the UV–Vis absorption and luminescence spectra in aqueous solution. The addition of increasing amounts of DNA (in the range 0.0 to 17.9 μM) to the ZnTRP solution led to a pronounced hypochromic effect on the Soret band at 426 nm (Fig. 2), in parallel with a bathochromic shift to 438 nm. However, when the concentration of DNA reached the 17.9 to 127 μM range, the Soret band remained at 438 nm and only a

Conclusions

ZnTRP is a promising PDT sensitizer, that acts as a Type II photooxidation catalyst for DNA, generating O₂(¹Δ_g) in the presence of light and dioxygen. The Zn ion coordinated to the porphyrin ring can play an additional role, by promoting a strong association with DNA via electrostatic and/or coordination interactions with the phosphate groups. The kinetics of the photooxidation reaction is consistent with the direct attack of the photogenerated O₂ (¹Δ_g) species, to dGuo bases.

Acknowledgements

The support from the FAPESP, CNPq and PRONEX (Brazil), and the J.S. Guggenheim Memorial Foundation (H.E.T.) is gratefully acknowledged.

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Zinc tetraruthenated porphyrin binding and photoinduced oxidation of calf-thymus DNA

Abstract

Introduction

Section snippets

Chemical

Fluorescence and electronic absorption measurements

Interaction of ZnTRP with DNA

Conclusions

Acknowledgements

Biochim. Biophys. Acta Gene Struct. Expr.

J. Inorg. Biochem.

J. Photochem. Photobiol. A

J. Photochem. Photobiol.

Photochem. Photobiol.

J. Phys. Chem. A

Inorg. Chem.

Biochemistry

J. Phys. Chem. B

Photochem. Photobiol.

J. Am. Chem. Soc.

J. Biomol. Struct. Dyn.

Nucleic Acids Res.

Biochemistry