Interaction of tamoxifen citrate with salmon sperm DNA
and bovine serum albumin
Zhu Ling, Liu Xiaoyan, Zhang Haixia
(State Key Laboratory of Applied Organic Chemistry, Lanzhou University, LanZhou 730000) Abstract: Interaction of tamoxifen citrate (TC) with salmon sperm DNA and bovine serum albumin (BSA) were investigated by various spectroscopic analysis methods under simulative physiological conditions. Thermodynamic parameters showed that the interaction process was spontaneous and hydrophobic forces play a major role in the binding between BSA and TC. The quenching constant KSV, the bimolecular quenching constant kq and the numbers of binding sites n (about 1) of both DNA and BSA to TC were similar. The value of n approximately equal to 1 indicated that there is only one type of binding site for DNA or BSA to TC. However, the binding constant (K) of DNA to TC was lower than that of BSA.
Keywords:Tamoxifen citrate;salmon sperm DNA;bovine serum albumin;spectroscopic
hyletylamine and has been the clinical choice as adjuvant or additional therapy following primary treatment for early stage breast cancer [1-3]. It belongs to the selective oestrogen receptors modulator (SERM) family with high affinity and selectivity to the oestrogen receptor (ER)
resulting in molecular and biological actions .
图 1 枸橼酸他莫昔芬的结构 Fig.1 Structure of tamoxifen citrate
DNA is the primary holder of genetic information for cells. The interaction between DNA and small molecules has attracted continuous interests in chemistry and biology because these interactions may influence replication and transcription of DNA and cause gene mutations leading
to some diseases . The interaction is not only conducive to the exploration and development of new nucleic acid probe, but contributes to investigate the molecular mechanism of drugs and
clarify the effect such as carcinogenic toxic and teratogenic molecular biology incentives, which further provides theoretical guidance for designing of more effective clinical drugs.Many studies have been devoted to the interaction between DNA and the drug [6-10].
Bovine serum albumin (BSA) has similar structure with human serum albumin (HSA) [11-15], which is one of most used model proteins for studying the interaction of protein and small molecule because it plays important roles in the transport, distribution and metabolism of many exogenous ligands, such as fatty acids, amino acids, drugs and pharmaceuticals [16-19].
Protein-drug interaction has significance in pharmacology, which can affect the biological activity
Foundations: The National Natural Science Foundation of China Fund (No.20775029, J0730425);The Program for New Century Excellent Talents in University (NCET-07-0400);The Central Teacher Plan of Lanzhou University
Brief author introduction:朱玲；女；硕士研究生；分离科学
Correspondance author: 张海霞；女；教授博士生导师 色谱分析在生物样品中的应用、治疗糖尿病中药的
提取分离及药理研究和构效关系. E-mail: firstname.lastname@example.org
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[20, 21] and toxicity [22-24] of drug.
No study had reported on the interaction between DNA or BSA and tamoxifen citrate (TC). This study aims to find the binding mode, binding constant K and the number of binding site n in
the systems of DNA or BSA and TC and further compare each other under simulated physiological conditions by UV-Vis spectroscopy, fluorescence spectroscopy, circular dichroism
and other methods .
1.1 Chemicals and Apparatus
BSA (fatty acid free) was obtained from Shanghai lizhudongfeng Biochemical Technology Co.Ltd. (Shanghai, China). Salmon sperm DNA was purchased from Shanghai Chaoyan Biological Technology Co., Ltd. (Shanghai, China). TC was purchased from Suzhou Sunary Pharmaceutical Co., Ltd. (Suzhou, China). Other chemicals were of analytical grade and the double-distilled water was used throughout the experiments.
The absorption spectra were recorded on a Lambda 35 spectrophotometer (PerkinElmer, USA) equipped with 1.0 cm path length quartz cells. Fluorescence measurements were recorded on a RF-5301PC Spectrofluorophotometer (Shimadzu, Japan) using 10/10 nm bandwidths. The excitation wavelength of BSA was 290 nm, and the emission was read at 300-500 nm. The excitation and emission wavelength of TC is 277 nm and 200-350 nm, respectively. Circular dichroism spectra were measured with an Olis DSM-1000 Circular Dichroism Spectrometer (Olis, USA) at room temperature over a wavelength range of 170-450 nm and under nitrogen flush.
1.2 Solutions and methods
Stock solutions of BSA and DNA were prepared in buffer solution and kept in the dark at 277 K. The concentration of DNA was determined according to the absorbance at 260 nm after establishing that the absorbance ratio A/Awas in the range 1.80-1.90 . The molarities of 260280 ?1?1 -3 DNA were calculated using ε=6600 Mcm. The buffer used consists of Tris (5×10mol DNA-1-1L) and NaCl (0.05 mol L), and adjusted the pH to 7.20 with HCl.
-3 -1Stock solution of TC (1.0×10mol L) was prepared in DMSO because of its poor solubility
in water for the UV-Vis and fluorescence titration experiment.
To study the interaction between DNA and TC, the concentration of TC was fixed with the changed concentration of salmon sperm DNA in fluorescence and UV experiments.
To study the interaction between BSA and TC, the concentration of BSA was fixed with different drug concentration. The fluorescence titration experiment was accomplished as following:
-5 ?13.0 mL BSA solution at concentration of 1.0×10mol L, was titrated by successive additions of ?3 ?110 μL stock solution of TC (1.1×10mol L) with a micro-injector manually. The mixture was
allowed to stand for 3 min. The fluorescence spectra were then measured at four temperatures (288, 296, 303 and 310 K).
2 Results and discussion
2.1 The interaction between DNA and TC
2.1.1 UV-vis absorption studies
The UV-vis absorption spectra of TC are recorded with the titration by salmon sperm DNA in Fig. 2. DNA has two peaks at 204 nm and 259 nm, however, the absorption is much lower than that of TC with similar concentration. TC shows a little decrease in the peak intensity
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(hypochromic effect) with the increasing concentration of DNA. Slightly red shift is observed in
the UV spectra, which represents that the binding mode of TC to DNA is intercalative binding.
图 2 DNA-TC 的紫外吸收光谱 -6 -5 Fig.2 UV absorption spectra of TC-DNA, C=2.2×10mol/L, Cfrom (1-4):0, 1.32, 1.76, 2.20 (×10mol/L), TCDNA -4C=1.90×10mol/L (5) DNA
2.1.2 Fluorescence spectroscopy studies
The fluorescence spectra of TC in the presence of different concentrations of DNA are shown in Fig. 3. With the increasing concentration of salmon sperm DNA, the fluorescence intensity of tamoxifen citrate decreased remarkably, with no shifts at the maximal emission.
图 3 不同浓度的 ss-DNA 对 TC 的荧光光谱图 -6Fig.3 Fluoresence quenching spectra of TC at different concentrations of ss-DNA, λ= 277nm.C=5.5×10 ex TC -6 mol/L; Cfrom (1-8): 0, 1.52, 3.04, 4.56, 6.08, 7.60, 9.12, 10.64 (×10mol/L). DNA
The obvious quenching to fluorescence of TC after adding salmon sperm DNA showed that TC acted on the DNA bases and the presence of salmon sperm DNA resulted in the change of microenvironment of TC. That is to say, TC can be used as the probe of salmon sperm DNA . According to the Stern-Volmer equation :
F 0 = 1 + kqτ [Q] = 1 + KSV [Q] (1)F
Where Fand F are the fluorescence intensities before and after addition of DNA, 0
respectively, [Q] is the concentration of salmon sperm DNA, kis the bimolecular quenching q ?1?1?1constant (k: L mols), Kis the Stern-Volmer quenching constant (K: L mol), τ is the qSV SV-8 lifetime of the fluorophore, as to reference , τ=10s.
The Stern-Volmer quenching plot of F/F with concentration of DNA is linear and it is easy 04 ?1 to calculate the quenching constant Kas 3.06×10L moland the bimolecular quenching SV 12 ?1?1constant kq as 3.06×10L mols,which is larger than the limiting diffusion rate constant of 10 ?1?1biomolecule (2.00×10L mols) and indicates the static quenching occurred in the system of
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TC quenching by DNA [32,33].
In order to evaluate the binding constant (K) and the number of binding sites (n), the
following equation  was applied:
( F 0 ? F ) (2) log[ ] = log K + n log[Q]F 4 ?1?1K is calculated as 1.64×10L mols, n is 0.94. The value of n approximately equal to 1
indicating that there is only one type of binding site for DNA to TC.
2.1.3 Circular dichroism spectra
Circular dichroism (CD) is a useful technique to monitor changes in the DNA morphology
and subtle variations of DNA chiral conformation .
Because of its right-handed helicity, the CD of salmon sperm DNA (soild line in Fig. 4) shows a positive band centered at 275 nm and a negative band centered at 245 nm. Adding TC in DNA and kept for 12 hours, it can observe a decrease of the intensity and a red shift of the positive and negative CD band of DNA, which suggested the DNA structural change.
图 4 DNA 和 TC-DNA 的圆二光谱图 -5 -1-5 -1Fig.4 CD spectra of Salmon sperm DNA in the presence of TC. C=1.80×10mol L, C=3.83×10mol L. DNATC
2.2 Interaction between BSA and TC
2.2.1 UV-vis absorption studies
The UV-vis absorption spectra of BSA and its UV titration with TC are recorded in Fig. 5.
The absorbance of TC is much higher than BSA with similar concentration. However, the peak of BSA at 203 nm decreases and red-shift acutely with the adding of TC. The phenomena indicated that the binding between BSA and TC may lead the major conformation of protein to reconstruct
into α-helix and it also made the microenvironment of protein hydrophobic simultaneously .
图 5 BSA-TC 的紫外吸收光谱图 -6 -6 Fig.5 UV absorption spectra of BSA-TC, C=2.2×10mol/L (1), C=1.00×10mol/L,Cfrom (2-5): 0, 3.67, TCBSATC -6 7.33, 11.0 (×10mol/L).
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