Study On The Function And Application Of The Interaction Between Biomolecule And Micromolecule By Multi-spectral Methods

Objective:The essential biological materials, serum abumin and DNA, have been playing many vital roles for all kinds of biological phenomena. Protein-micromolecule and DNA-micromolecule interactions have a great influence upon the distribution of the micromolecules in the body, upon their patterns of metabolism and excretion. Investigating the binding mechanisms can provide an importance theoretical base on discussing human disease mechanism, diagnosis and prevention of diseases, pharmaceutical industry and development new drugs. Exploring the interaction mechanisms on theseSpectroscopy is always used to study the interaction between serum albumin and small molecules, DNA and small molecules. The interaction between serum protein and small molecules, DNA and small molecules by fluorescence quenching spectroscopy obtained under different temperature can give us a lot of information from the molecular level, such as quenching type, binding constant, binding sites, thermodynamic parameters and nature of the binding forces. Spectroscopy is an important method to study the interaction between serum albumin and small molecules, DNA and small molecules.This paper consists of the following two parts:1. Multi-spectral method is used to investigate the interaction between bovine serum albumin and erucic acid. Serum albumin, the major soluble protein, constituents of the circulatory system, is the most important drug carrier protein with a high concentration in blood plasma and has many physiological functions. It can bind many exogenous and endogenous ligands in blood, and realize transport and distribution of many molecules and metabolites. Erucic acid is a fatty acid in rapeseed oil, and excessive intake of it may contribute to heart damage. Therefore, the research of the interaction between erucic acid and bovine serum albumin can provide useful information in molecular level for erucic acid, which gives a comprehensive exposition of erucic acid in the human body, and contribute to the further explanation of the contact between heart disease and the erucic acid.2. Spectroscopy is used to study the interaction between double-stranded DNA (dsDNA) and unmodified nanoparticles (AuNPs). It has been widely accepted that single-stranded DNA (ssDNA) bind on unmodified AuNPs through electrostatic adsorption, but dsDNA and unmodified AuNPs repel each other through electrostatic repulsion. Based on the principle researchers establish colorimetric detection of DNA sequences. However, the researchers later found that the ssDNA adsorb on AuNPs through hydrophobic forces, rather than electrostatic force, so we suspect that the interaction between dsDNA and AuNPs is not electrostatic forces. But so far, there has been no research on the interaction between the two. Therefore, this paper makes a deep study on the interaction between the dsDNA and AuNPs, and discusses the interaction force type of the two, which is beneficial to the further study of the AuNPs and DNA.Method:1. Firstly, fluorescence spectrometry under different temperatures (298,304 and 310 K.) is studied between bovine serum albumin and erucic acid under simulated physiological conditions, and we can get information of the quenching constants, number of binding sites and so on; at the same time, through the Van’t Hoff equation of thermodynamic constants we can get force type; the ultraviolet spectroscopy is also used to the study of quenching type; synchronous fluorescence spectroscopy (△λ=60 nm), circular dichroism spectroscopy, fourier transform infrared spectroscopy are used to study the bovine serum albumin secondary structure changes after adding erucic acid; molecular simulation method studies the docking between bovine serum albumin and erucic acid. Various methods studies the interaction of bovine serum albumin and erucic acid from different angles, and they support each other. The characteristics of each method comprehensively reflects the interaction of erucic acid and bovine serum albumin, which makes contribution for the interaction between the erucic acid and heart disease.2. The interaction between dsDNA and AuNPs is studied by multi-spectral method. It is proved that the dsDNA could be adsorbed on the gold nanoparticles.Firstly, the fluorescence intensity of rhodamine B (RB) in the following four groups is first studied:RB solution, RB and dsDNA mixed solution, RB and AuNPs mixed solution, RB, dsDNA and AuNPs mixed solution. Then the fluorescence intensity changes of the DNA before and after adding AuNPs is studied by dsDNA tagerted with rhodamine green. The interaction of AuNPs and dsDNA is discussed through the changes of the fluorescence in the above two groups. The fluorescence titration experiments at different temperatures (308,313 and 318 K) is studied, and the Van’t Hoff equation explains why the binding force between dsDNA and AuNPs is weaker than that of ssDNA. Therefore, based on the principle of the adsorption of AuNPs by dsDNA, the principle of the color change of AuNPs under salt solution, the rapid colorimetric method was established for the detection of dsDNA. The allele-specific amplification PCR products of HT29, Ec109, A549, SW480 and Huh-7 cells was used to detected V600E BRAF mutation by colorimetric method.Result:1. This paper studies the interaction between bovine serum albumin and erucic acid. Study on fluorescence spectra shows that the intrinsic fluorescence of bovine serum albumin is quenched by erucic acid. The quenching constants of 298,304 and 310 K were 2.27x 105M-1,2.55×105 M-0 and 8.30 x 105M-1, respectively. With the increase of temperature, the quenching constant becomes larger and larger, which is a typical characteristic of the dynamic quenching. The calculated diffusion collision quenching constants Kq is greater than 1013 L·mol-1·S-1 (the maximum diffusive collision quenching constants of dynamic quenching are less than 2.0 x 1010 L·mol-1·S-1), which is a characteristics data for the static quenching. In order to verify the quenching type, ultraviolet spectroscopy is applied. Ultraviolet spectroscopy corroborates that the interaction between erucic acid and bovine serum albumin is dynamic quenching. Through the above experiments, it is concluded that the quenching method is a dynamic quenching method. The binding sites of 298,304 and 310 K are 0.89,0.95 and 0.92, respectively, so there is only one binding site for the bovine serum albumin and erucic acid. The binding distance r 2.76 nm, is less than 7 nm. Through the determination of the thermodynamic constants under different temperature, △H, AS are caculated to be 119.14 kJ/mol and 488.89 J/mol·K, respectively. △H>0, △S>0 indicate that the interaction between the two is mainly hydrophobic interaction. By means of synchronous fluorescence method, circular dichroism spectroscopy and fourier transform infrared spectroscopy, the structure of the secondary structure of protein is studied. △λ=60 nm of synchronous fluorescence spectrometry results show that the λmax of bovine serum albumin shift from 338 nm to 335 nm after adding the erucic acid, which is a proof of the decrease of tryptophan microenvironment polarity. Circular dichroism spectroscopy results show that the a-helix of the bovine serum albumin increases from 36.51% to 37.37% after the addition of erucic acid. Erucic acid changes the secondary structure of bovine serum albumin, which causes the a-helix structure more stable. After the addition of erucic acid, fourier transform infrared spectroscopy results show that a-helix increases from 11.83% to 14.06%, β-angle droppes from 46.27% to 29.82% and β-sheet increases from 30.78% to 41.93%, which also prove that the secondary structure of bovine serum albumin is changed by adding erucic acid. The results of circular dichroism spectroscopy and fourier transform infrared spectroscopy both prove that the addition of erucic acid inecrease the amount of a-helix. Molecular simulation results are in good agreement with the experimental findings, which proves that erucic acid binding to bovine serum albumin subdomain IIA in the hydrophobic cavity, and the main force is hydrophobic interaction and hydrogen bonding. Erucic acid docks with position near Tyr-149, Tyr-187, Tyr-451, Val-240, Val-188, Leu-237 and His-241 residues.2. We find that dsDNA can be adsorbed on AuNPs, and the binding force between them is proved by fluorescence spectroscopy.Fluorescence spectroscopy experiments find that dsDNA could be adsorbed on the surface of AuNPs and reduce free dsDNA and AuNPs in solution, so RB combines less dsDNA and AuNPs, resulting in changes in the fluorescence of RB. At the same time, the fluorescence intensity of dsDNA (labeled with rhodamine green) decreases after the addition of the AuNPs, and it is also proved that the dsDNA is adsorbed on the AuNPs surface. So we prove that the two groups of experiments both prove that the dsDNA can be adsorbed on the AuNPs. Fluorescence titration experiments results under different temperatures (308,313 and 318 K) are caculated. A positive AS value is frequently considered evidence for hydrophobic interactions. Moreover, a positive AS and a negative △H value represent electrostatic interactions. △H is-132.33 J/mol, and △S is 12.46 J/mol·K, which represents the presence of electrostatic force and electrostatic interactions. Therefore, the results show that interaction between the two is the result of the hydrophobic interaction and electrostatic interaction. Hydrophobic force is attractive force and electrostatic force is mutual repulsion force. Due to the presence of electrostatic force, the adsorption capacity of dsDNA onto AuNPs is weaker than that of ssDNA. Due to the dsDNA protect AuNPs from aggregation in high salt concentration, so the AuNPs color keeps red; without dsDNA protection, unmodified AuNPs color under high salt condition becomes blue. As a result, we established a colorimetric detection method based on the interaction of AuNPs and dsDNA. This experiment observates results with naked eye, and need no additional equipment. The method is simple and fast, and the detection process is not more than 15 min.The BRAF V600E point mutation was successfully detected by the principle of PCR and dsDNA/unmodified AuNPs. In the experiment, we first design the primers of allele specific PCR, so that only the mutant type can get a large amount of DNA, while the wild type can not be amplified. The amount dsDNA of wild type and mutant type present an larger differences, resulting the aggregation state differences in AuNPs at high salt concentration that the naked eyes can observe. The mutant gene in HT29,Ec109, A549 and Huh-7 cells are rapidly detected by the dsDNA/AuNPs method. There was no false positive and false negative results in the experiment, and there is no need to purify the PCR product. The method is simple and easy to operate, and the result is stable. Compared with ssDNA/unmodified AuNPs method, it is easier to be applied for actual sample detection.Conclusion:The interaction between biological molecules and small molecules is studied by spectroscopy mainly on the following two aspects:on the one hand, it is used to study the interaction between protein and erucic acid. It makes a contribution to erucic acid basic research, and to reveal the erucic acid metabolism dynamics in vivo. It has the vital significance to study the relationship between erucic acid and heart disease. On the other hand, it is used to study the interaction between dsDNA and AuNPs, which demonstrate that the dsDNA can be adsorbed on the AuNPs. On the performance of this we establishes a rapid colorimetric detection method, and the method requires no additional instruments. BRAF V600E mutation was successfully detected using this new technique. Spectroscopy plays a very important role in biological molecules and small molecules interaction study. We study the interaction of dsDNA and AuNPs, which is a supplement principle of mutual effect between the DNA and AuNPs. This method lay the foundation of AuNPs and DNA detection. In addition to BRAF V600E mutation, the technique can be used in other point mutations.