Study of Photoinduced Interaction between Calf Thymus-DNA and Bovine Serum Albumin Protein with H 2 Ti 3 O 7 Nanotubes

Hydrogen titanate nanotubes were synthesized by hydrothermal process using 10 M NaOH and TiO2 anatase powder. The material synthesized was characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to ensure the structural and morphological characteristics. The interaction of calf thymus DNA (CT-DNA) and bovine serum albumin protein with suspended aqueous solution of titanate nanotubes was investigated using UV absorption spectroscopy and the apparent association constant was found to be, Kb= 1.68 × 10 M and Kap=5.41 × 10 M for DNA and BSA respectively. Addition of the titanate nano material resulted quenching of fluorescence spectra of ethidium bromide-DNA in tris HCl buffer solution and that of aqueous protein solution. The apparent binding constant (Ksv= 5.46 × 10 M for DNA binding and Ksv = 6.063 × 10 M for protein binding) was deduced from relevant fluorescence quenching data using Stern-Volmer equation.


Introduction
One-dimensional nanostructures have drawn a great attention due to their potential applications in a variety of novel devices in recent past [1][2][3][4][5].The most fascinating example of such nanostructure is of course carbon nanotubes.Several efforts were made to synthesize nanorods and nanowires of more complex structure and conesquently a number of one-dimensional nanomaterials including metals, oxides and nitrides have also been reported.Hydrothermal or solvothermal synthesis is an very important approach to produce oxidic nanowires and nanotubes because synthesis of these types provide access to uniform and distinct morphologies in large scales with remarkable reliability, selectivity, and efficiency [6].
Among the one-dimensional oxidic nanomaterials reported, titanium oxide is of particular interest for its wide applications as catalyst supports, semiconductor photocatalysts and sensors [7][8][9].TiO 2 nanoparticles are acting as biosensors in chemical and biochemical fields and their applications are becoming more extensive.These probes have been applied to the ultrasensitive detection of proteins, DNA sequencing, clinical diagnostics, etc. TiO 2 nanoparticles have also been used as carriers for photosensitizer like porphyrins in photodynamic therapy for cancer treatment.Titanate nanotubes of nearly 8 nm in diameter were first reported by Kasuga and co-workers, employing a hydrothermal treatment of rutile TiO 2 powders in strong aqueous solution of NaOH at 110˚C followed by HCl washing [10].Several number of papers were published afterwards on the structure of hydrothermally synthesized TiO 2 which also gave birth of a number controversies.Finally Chen et al. concluded that these nanotubes were of H 2 Ti 3 O 7 structure based on diffraction and high-resolution transmission electron microscopy (HRTEM) results [11].
Over the past decade a significant number of papers have been published on the specific interaction between photoactive nanocrystalline metal oxide semiconductor particles and DNA or proteins or other biomolecules [12][13][14][15][16][17][18].Interestingly research works of these types open up the possibility of the electronic transduction of DNA sequence and hybridization, as well as development of new electrochemical probes for DNA-binding proteins, both of which are major challenges in bioelectronics [16,19,20] [22].It is also used as a nutrient in cell and microbial culture.BSA is used to stabilize some enzymes during digestion of DNA and to prevent adhesion of the enzyme to reaction tubes.This protein does not affect other enzymes that do not need it for stabilization.The binding properties of BSA with various drugs have been fully investigated by many researchers [23][24][25].In this present work, interaction between suspended H 2 Ti 3 O 7 nanotubes (in aqueous solution) and CT-DNA, followed by BSA protein and was examined by UV and fluorescence spectroscopy.As the natural fluorescence intensity of DNA is very weak, ethidium bromide (EB) was used to enhance the fluorescence intensity while BSA shows intrinsic fluorescence due to the presence of aromatic amino-acid residues.

Materials
CT-DNA and TiO 2 (AR grade) was purchased from Merck (Mumbai, India) while ehidium bromide and bovine serum albumin protein was purchased from Sigma Aldrich.Approximately 10 -4 M stock solution of CT-DNA was prepared by dissolving proper amount of DNA in 1 M tris buffer.

Preparation of\Hydrogen Titanate Nanotubes (HTO)
A 1.0 g potion of anatase TiO 2 powder was dissolved in 10M NaOH solution and the resulting suspension was then transferred in an autoclavable reaction chamber.The reaction chamber was autoclaved in an oven at a temperature of 130˚C for 22 hours.The product was filtered after cooling down to room temperature and thoroughly washed with 1 M HCl and water to remove any trace of NaCl produced during washing [26].Subsequently the material was dried at ambient temperature.

Characterization of HTO
The powder X-ray diffraction (XRD) data were recorded from a PANalytical X'pert Pro diffractometer with Cu Kα radiation.The morphology of the nanostructured samples was studied with use of a Jeol JSM-840 scanning electron microscope (SEM) operating at 120 keV.TEM studies of nanotubes were carried out with a FEI Tecnai TEM equipped with a LaB 6 filament and operated at 200 kV.TEM samples were prepared by placing a drop of the ultrasonically dispersed powder (in ethanol) on a carbon-coated copper grid and drying in air.

Fluorescence Quenching Measurements
A Hitachi F-4500 fluorescence spectrophotometer was used to perform the fluorescence quenching measurements.The excitation wave length of EB-DNA was kept fixed at 522.0 nm while the emission wave length was chosen at 400.0 nm.The excitation and emission slit length (5 nm and 10 nm respectively) wave lengths as well as the scan speed (2400 nm/min) were kept fixed throughout the experiment.For BSA-HTO interaction study the excitation and emission wavelength was kept fixed at 510 nm and 435 nm respectively with all the other conditions remaining the same as before.

Absorption Spectral Measurements
To carry out the absorption spectral measurements, a JASCO V-570 UV-Vis spectrophotometer was used.For each measurement, 4.0 mL solution, containing DNA, suspended solution of nanoparticle and tris buffer was taken.At first the UV-vis spectrum was recorded for a solution without DNA.For the rest of the solutions, DNA was added with an increment of 0.05 mL each and then the spectra for all the solutions were recorded in the range of 200 -800 nm.BSA absorbs light energy around 280 nm.So the absorption spectra for purely aqueous solution of BSA (10 -2 M) was recorded at the beginning and then suspended aqueous solution of HTO was added.

Structural and Morphological Studies of HTO
X-ray diffraction pattern of HTO is shown in Figure 1 This diffraction pattern mostly matches with the monoclinic crystal structure of H 2 Ti 3 O 7 (space group C2/m (12)) (JCPDS Card no.41-0192).Figure 2 shows the  SEM images of the nanostructured samples hydrothermally synthesized from anatase powder.The scanning electron micrograph shows the formation of one dimensional (1-D) nanostructures.Further TEM study (Figure 3) clearly shows that the each 1-D units are actually hollow, multiwalled nanotubes with inner diameter of 4 -5 nm and the outer diameter of 9 -10 nm.

UV Absorption Study by CT DNA and BSA with HTO
Figure 4 shows the absorption spectra of CT-DNA in presence of suspended aqueous solution of HTO.The concentration of suspended HTO was kept constant for all sets of experiments while the concentration of DNA was increased uniformly.In presence of HTO the absorbance of DNA increases considerably with increasing concentration of DNA.Similar type of experiment was  performed with BSA and HTO and the only difference is that the concentration of BSA was kept constant throughout the experiment.Figure 5 shows the gradual increase of absorption spectra with increasing concentration of HTO.To make it confirm that the absorption change which we observed in the spectrum was not due the experimental error, baseline corrections were done for all the measurements.Therefore the results clearly indicate that there is a interaction between suspended HTO and CT DNA/BSA through the formation of some complex of the type HTO 7 DNA, HTO-BSA [27,28].

Fluorescence Quenching Study of EB-CT DNA by HTO
As it has been mentioned earlier that the natural fluores-cence intensity of DNA is very weak, ethidium bromide (EB) was used as fluorescence probe for DNA. Figure 6 shows that addition of suspended aqueous solution of HTO to a solution of EB-DNA resulted in a decrease in fluorescence emission intensity of EB-DNA.HTO solution was added in increasing concentration keeping the EB-DNA concentration constant for all the measurements.Fluorescence quenching study of BSA was carried out in similar fashion keeping the concentration of BSA solution fixed.Figure 7 shows the quenching of   BSA protein with increasing concentration of HTO.
Baseline correction was done for all the measurements to get rid of the probable scattering effect due to the suspended nature of HTO in aqueous medium.It ensures that fluorescence quenching occurs only due to interacttion between CT DNA/BSA and HTO.

Determination of Binding Constant between CT DNA and HTO
In order to further illustrate the binding strength of the HTO with CT-DNA, the intrinsic binding constant K b was determined from the spectral titration data using the following equation:  8), the binding constant K b for the HTO-DNA complex was determined to be 1.68 × 10 4 M -1 (R = 0.97208 for four points).
Fluorescence intensity of EB bound to DNA at 522 nm shows a decreasing trend with the increasing concentration of the HTO (Figure 6).The quenching of EB bound to DNA by the titanate nanorods are in agreement with the linear Stern-Volmer equation: where I 0 and I represent the fluorescence intensities in absence and presence of quencher, respectively.K sv is a linear Stern-Volmer quenching constant, Q is the concentration of quencher.In the quenching plot (Figure 9) of I 0 /I versus [complex], K sv value is given by the ratio of the slope to intercept.The K sv value for the complex is 5.46 × 10 4 (R = 0.96771 for four points), suggesting a strong affinity of the titanate nanorods to CT-DNA.Again the apparent binding constant (K ap ) due to interaction between HTO with BSA can be determined following the equation: 6.0×10 -6 8.0×10 -6 1.0×10 -6 1.2×10 -6 1.4×10 -6 1.6×10 -6 1.8×10 -6 2.0×10 -6 2.2×10 -6 [DNA] 7.2×10 -9 7.0×10 -9 6.8×10 -9 6.6×10 -9 6.4×10 -9 6.2×10 -9 6.0×10 -9 5.8×10 -9 [DNA]/(ε a -ε where A 0 and A c are the absorbance of BSA and complex respectively at 280 nm and Aobs is the observed absorbance of the solution containing different concentrations of suspended HTO at 280 nm.From the plot of log[A 0 -A]/A vs 1/[HTO] (Figure 10) for the absorption titration of BSA with the HTO the K ap was determined to be 5.41 × 10 3 (R = 0.9558 for four points).Fluorescence intensity of BSA at 280 nm shows the similar trend as the DNA which is mentioned earlier and the binding constant can be determined by Stern-Volmer equation as usual.In the quenching plot (Figure 11) of I 0 /I versus [HTO] for BSA-HTO interaction, K sv value is given by the ratio of the slope to intercept.The K sv value for the complex is 6.063 × 10 3 (R = 0.9953 for four points).The binding constant values for both cases (K ap and K sv ) indicate a considerably strong affinity of the HTO to BSA.

Conclusion
Study of photoinduced interaction between HTO and CT DNA using UV and fluorescence spectroscopic tools established that the interaction is quite strong which is also revealed by the high binding constant.The present work may be helpful in understanding the interaction between various photosensitizers and DNA.Clinical application of the existing photosensitizer is associated with some problems such as aggregation of photosensitizers in aqueous medium, decrease in quantum yield due to aggregation, toxicity etc. HTO nano particles can overcome all the above mentioned problems.This observation can also be considered with importance to investigate such interaction between nanotubes and DNA for future application in photodynamic therapy.This study of interaction of HTO with physiologically important BSA protein will provide some necessary information for the design and application of new drugs.ultrasensitive detection of proteins, clinical diagnostics etc.
where[DNA]  is the concentration of DNA,  f ,  a and  b correspond to the extinction coefficient, respectively, for the free HTO, for each addition of DNA to the suspended solution of HTO and for the HTO-DNA complex in the fully bound form.A plot of  , gives K b , the intrinsic binding constant as the ratio of slope to the intercept.From the 

Figure 8 .Figure 9 .
Figure 8. Plot of [DNA]/( a −  f ) vs [DNA] for the absorption titration of CT-DNA with the HTO in Tris-HCl buffer.

Figure 10 .Figure 11 .
Figure 10.Plot of log[A 0 -A]/A vs 1/ [HTO] for the absorption titration of BSA with the HTO in aqueous medium.
. Deoxyribonucleic acid (DNA) is unique genetic molecule in any living organism.Consequently the qualitative and quantitative analysis of nucleic acids is Study of Photoinduced Interaction between Calf Thymus-DNA and Bovine Serum Albumin Protein with H 2 Ti 3 O 7 Nanotubes