Elucidation of the Binding Properties of A Photosensitizer to Salmon Sperm DNA and Its Photobleaching Processes by Spectroscopic Methods
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- Zhang, L. & Tang, GQ. J Fluoresc (2013) 23: 303. doi:10.1007/s10895-012-1148-8
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Methylene blue (MB) is a tricyclic heteroaromatic photosensitizer with a promising application in the photodynamic therapy (PDT) for anticancer treatment. The binding properties of MB to salmon sperm DNA have been investigated by the measurements of absorption spectra, quenching experiments and the photobleaching processes. Remarkable hypochromic and bathochromic effects of MB in the presence of increasing amounts of DNA have been observed in the absorption spectra. The quenching of MB by the DNA bases obeys the Stern-Volmer equation and ferrocyanide quenching of MB in the absence and presence of DNA is also measured as extended experiments. Results from the above spectral measurements are all consistent with the intercalative binding mode of MB to DNA with the Kb value of 5.6 × 103 M−1. The photobleaching processes of MB and its DNA complex have also been studied, which indicate that the photobleaching of MB and its DNA complex proceed with different mechanisms and the reactive oxygen species are responsible for the self-sensitized photooxidation of MB.
In recent years, there has been a growing interest in the study of various small organic molecules-DNA interactions because of its importance in understanding the drug-DNA interactions and the consequent design of new efficient drugs targeted to DNA [1–5]. It is generally accepted that three models about binding of small molecules to the DNA double helix have been identified: intercalative binding, groove (or surface) binding and electrostatic binding [6, 7]. Electrostatic binding interactions between cationic species and the negatively charged DNA phosphate backbone usually occur along the exterior of the helix. Groove binding generally involves direct hydrogen-bonding or van der Waals interactions with the nucleic acid bases in the deep major groove or the wide shallow minor groove of the DNA helix. Stacking interactions between nucleobases and aromatic ligands are important in defining the third type of binding mode known as the intercalative binding, which is defined when a planar, heteroaromatic moiety slides between the DNA base pairs and binds perpendicular to the helix axis. Therefore, it is apparent that the intercalative binding and groove binding are related to the grooves in the DNA double helix but the electrostatic binding can take place out of the groove.
All the chemicals used in this work were of analytical reagent and used without further purification, unless otherwise stated. Double distilled water was used for solution preparation. Commercially prepared salmon sperm DNA was obtained from Sigma Chemical Co. and stored at 4 °C. To prepare stock solution, it was directly dissolved in water at a DNA concentration of 0.5 mmol/L in nucleotide phosphate, the concentration of which had been determined by absorption spectroscopy as described in the literature methods [29, 30]. The stock solutions of the photosensitizer MB and the quencher K4Fe(CN)6 were both 0.1 mmol/L in concentration, and stored in dark. The buffer solution of pH 7.1 contains 0.5 mol/L NaCl and 0.05 mol/L Tris. All above solutions were further diluted as required. It should be noted that all the following spectroscopic measurements are performed in the aerated environment. We have also tested the influence of molecular oxygen on the absorption and emission spectra and found no variation of the above spectral features.
UV-Visible absorption spectra in aqueous solution by using a quartz cell having 1.0 cm pathway were all recorded on a computer-controlled JASCO V-570 spectrophotometer. 1.0 ml of buffer solution (pH 7.1) and 0.2 ml of MB solution were transferred to a 10 ml standard flask. A known volume (0.2, 0.4, 0.6, 0.8 ml) of DNA stock solution was also added. The above solutions were then mixed, diluted to the volume with water, and incubated for 5 min. Pure MB solution was prepared in a similar manner without DNA and buffer solution. The final concentration ratio of the dye to DNA (in nucleotide phosphate) ranges from 1:5 to 1:20 in this study.
All the emission spectra were measured on Acton Research SpectroPro-300i spectrometer with spectral CCD operating at −15 °C. Xenon arc lamp is used as the excitation light source in the measurements of emission spectra. Solution used in the measurement of emission spectra was prepared the same as the above measurement of absorption spectra. The excitation wavelength was set at 630 nm.
Ferrocyanide Quenching Measurements
The fluorescence quenching experiments with ferrocyanide (K4Fe(CN)6) were also performed. 1.0 ml of buffer solution (pH 7.1), a known volume (0.2 and 0.8 ml) of DNA stock solution, 0.2 ml of MB solution and 1.0 ml of ferrocyanide solution were transferred to a 10 ml standard flask. The above solution was mixed, diluted to the volume with water, and incubated for 5 min. Pure MB solution with the quencher ferrocyanide was prepared in a similar manner without DNA and buffer solution.
The photobleaching of the photosensitizer MB and its DNA complex as a result of the laser light irradiation was carried out in this work. Sealed in a tube, 50 μl of the MB solution (2 μmol/L) in the absence and presence of DNA (10 and 40 μmol/L) was irradiated with the instrument for PDT developed in our laboratory (670 nm semiconductor laser output, 35.0 mW laser power at the focal point). Every 5 min, the emission spectra of MB solution were measured by xenon light excitation (λex = 630 nm), while the laser irradiation was turned off at that moment.
Results and Discussion
Absorption and fluorescence properties of the photosensitizer MB change dramatically as a result of binding to DNA, and these changes have been quantified to estimate binding constants and sequence specificities and to evaluate the DNA binding mode.
A plot of [DNA]/Δεap versus [DNA] will have a slope of 1/Δε and a y-intercept equal to 1/(ΔεKb). Kb is then given by the ratio of the slope to intercept. Since a double-reciprocal plot gives excessive weight to data points obtained at low [DNA], the half-reciprocal plot should generally be more accurate. The change in the absorbance of MB with increasing DNA concentration was used to construct the half-reciprocal plot as shown in Fig. 2b. The half-reciprocal plot of the absorption titration data according to eq. (2) gave a linear plot and resulted in an intrinsic binding constant of 5.6 × 103 M−1 in DNA base pairs, which is consistent with the literature reports.
It should be noted that similar spectroscopic properties as mentioned above are occasionally observed for minor groove binders. Therefore, the binding mode of MB to salmon sperm DNA may be further questioned considering this point. We agree that the minor groove-binding mode may be probable for some organic molecules, but it should be unlikely for MB in this case. In support of our conclusions, a recent theoretical modeling study reveals a preference for symmetric intercalation of MB to DNA with an alternating GC base sequence, while asymmetric intercalation and minor and major groove binding appear to be less favorable . This result also matches with the published circular dichroism data . It is also worthy of note a few recent publications by Zhao et al., in which spectroscopic and electrochemical measurements of MB loaded β-cyclodextrin with DNA reveal that the binding model of confined MB to DNA is the electrostatic mode [27, 28]. Considering diverse systems used (MB loaded β-cyclodextrin with DNA versus MB directly complexed with DNA), it is easy to understand why different binding modes are finally concluded.
Ferrocyanide Quenching Measurements
In summary, the binding properties of photosensitizer MB to salmon sperm DNA have been studied by spectroscopic methods in this work. MB binds to double helical DNA with a high affinity. Remarkable hypochromic and bathochromic effects of MB in the presence of increasing amounts of DNA have been observed in the absorption spectra. The quenching of MB by the DNA bases obeys the Stern-Volmer equation and as further experiments, ferrocyanide quenching of MB in the absence and presence of DNA is also measured by fluorescence spectroscopy. Results from the above spectral measurements are all consistent with the intercalative binding mode of MB to DNA with the intrinsic binding constant (Kb) of 5.6 × 103 M−1. The photobleaching processes of MB and its DNA complex have also been investigated. On one hand, singlet oxygen is formed via energy transfer from triplet MB to molecular oxygen (process (i) in Fig. 7). On the other hand, MB withdraws one electron from DNA upon irradiation, which results in the formation of reduced MB radical that is the precursor of superoxide radical (process (ii) in Fig. 7). These reactive oxygen species are both contribute to the self-sensitized photooxidation of MB. Elucidation of the above photobleaching processes of MB helps us to obtain a better understanding of the MB-DNA interactions in vivo and may facilitate our future investigation of the photodynamic activities of MB in biological organisms.
This work was supported by the Fundamental Research Funds for the Central Universities.