Measuring Binding Constant of Ethidium Bromide (EtBr) to DNA

Measuring Binding Constant of Ethidium Bromide (EtBr) to DNAABSTRACTThe principle behind this lab try out was to determine the binding ceaseless of ethidium banality to DNA using the know concentrations and the calculated rank of X obtained from the equation. Ethidium bromide is said to have a blue affinity to DNA, therefore, the expected note value for the binding everlasting should be large. Two mode were implemented in determining the binding constant of EtBr to DNA. The first method was by inputting the absorbance data using a fixed excel worksheet. The main goal was to manipulate the value in cell I24 to be as low as possible. After several trails and error, the final value of cell I24 was 0.00011316 and logK was 3.99. The second method was by simple manual calculation. The twain methods retorted two very antithetic results. By manual calculation the obtained value for K was 37,108.63 M-1, which was apocalyptic that the final EB complex of DNA was larger than that of unbound DNA and unbound EB. The exalted value of the binding constant (K), demonstrates that there is a high affinity of ethidium bromide to DNA. This correlates to the expected values of EtBr, which are reported to be in between 104 M to 106 M.INTRODUCTIONDNA plays an important role in biological systems being that it contains hereditary materials that is passed on to generations after generations. DNA contains sequences of specific bases inside the DNA strands where it stores genetical information that can be readily replicated (Jeremy M. Berg, 2015). It is this sequence that determines the sequence of RNA and other protein molecules and it also transports most of the activities within the cells. RNA synthesis is a tell apart step in the expression of genetic information (Jeremy M. Berg, 2015, p. 859). DNA is more than just a source of sequence information, but it is also the platform where binding proteins collate. This is an important factor for the development of many cli nical drugs. The structure and function of drug proposes are the basis for designs of effective and specific inhibitors. Though to be considered effective, the target drugs must bind to the enzymes or receptors with great affinity and specificity.Ethidium bromide, (EtBr), is widely used in many scientific laboratories to study the binding properties of DNA. Ethidium bromide is an aromatic tinge that slips in between the base pairs of DNA. This binding of ethidium bromide to DNA is a process called intercalation. During this process, the structure of the DNA is changed and the distance among the base pairs in which it directly binds to increases. ensuant in the overall expansion of the dimension of DNA. Ethidium bromide exhibits diminutive base pair partiality containing moderate binding affinity depending on its ionic strength. The intercalation specificity depends greatly on inactive interactions and the formation of Van der Waals interactions between base pairs. It has the cap ability to bind itself to the hydrophobic interior in the stacked base pairs of DNA. However, Ethidium bromide is known to be a very potent mutagen, which is a chemical agent that can cause mutation in DNA cells and other trusted diseases such as cancer. It is called an intercalating agent due to its ability to inhibit cell growth, DNA replication and transcription.In this try out, we pass on be measuring the binding constant of the dye, ethidium bromide, using herring testes by method of absorption spectroscopy. Herring testes DNA (htDNA) is a natural DNA used in studies of DNA binding agents that tone DNA structure and function (Sigma-Aldrich, Inc). This method involves titrating a series of solutions varying in DNA concentrations but with a constant concentration of the intercalating agent, ethidium bromide. By implementing this set up of magnitude, it is likely to obtain samples with entirely unbound DNA and DNA that is saturated with ethidium. The solutions will run throug h the absorbance spectrometer at a wavelength of 480 nm. The absorbance measured will be used to calculate the bound ethidium. To determine the amount of bound ethidium the following expressions below is used, given that the information of D and E are known where E is the total ethidium bromide concentration and D is the total DNA concentration. This information will allow us to calculate the binding constant of ethidium toward DNA.The sense of equilibriumD + E CD = DNAE = Ethidium BromideC = ComplexK = C / DE(1)Solve for KK = x/E xD x(2)Kx2 x(KD + KE + 1) +KDE = 0 (3)Determination of the amount of bound ethidium (amount of complex, C)Aobs = b x + f E x(4)b (480 nm) = 2,497 M-1 cm-1f (480 nm) = 5,600 M-1 cm-1EXPERIMENTAL PROCEDURESMATERIALS2 mM DNA (bp) telephone circuit solution with BPES weaken2 M DNA (bp) stock solution with BPES buffer10 M stock solution of Ethidium BromideMicropipettesMicrocentrifuge tubesAbsorbance spectrometerPROCEDURESDetermine the volume of stock DN A solution and the amount of buffer needed for each of the nineteen samples before proceeding. *Refer to data tables*In the microcentrifuge tubes, make nineteen 1 mL DNA solutions by diluting from the 2 mM, and the 2 M DNA stock solutions with BPES buffer. Then add 10 L of ethidium bromide to the nineteen prepared solutions. prance well and measure the absorbance at 480 nm. Record the absorbance for each of the nineteen solutions and use the information to calculate the binding constant K.RESULTSDATA TABLEAbsorbance at 480 nm-LogbpDNA(bp)Volume 2 mM DNA Solution (in L)Volume 2 M DNA Solution (in L)Volume of BPES buffer (in L)0.0183.00.001500.0XXXXXX500.00.0233.35.01187-10-4251.0XXXXXX749.00.0243.71.99526-10-4100.0XXXXXX900.00.0324.00.000150.0XXXXXX950.00.0324.35.01187-10-525.0XXXXXX975.00.0294.71.99526-10-510.0XXXXXX990.00.0325.00.000015.0XXXXXX995.00.0315.35.01187 -10-63.0XXXXXX997.00.0305.71.99526-10-61.0XXXXXX999.00.0326.00.000001XXXXXX500.0500.00.0336.35.01187-10-7XXXXXX251.074 9.00.0336.72.51189-10-7XXXXXX100.0900.00.0327.00.0000001XXXXXX50.0950.00.0327.35.01187-10-8XXXXXX25.0975.00.0347.71.99526-10-8XXXXXX10.0990.00.0358.00.00000001XXXXXX5.0995.00.0358.35.01187-10-9XXXXXX2.5997.50.0338.72.51189-10-9XXXXXX1.0999.00.0309.00.000000001XXXXXX0.5999.5DATA GRAPH CALCULATIONS example Calculation for KUsing sample 4Known InformationAobs = b x + f E xAobs = 0.032b (480 nm) = 2,497 M-1 cm-1Einitial = 10 M = 1.0 -10-5 Mf (480 nm) = 5,600 M-1 cm-1DNA(bp)initial = log(bp) = -4.0 = 1.0 -10-4 MSolve for X Aobs = b x + f E x0.032 = (2497 M-1cm-1)x + (5600 M-1cm-1)(1.0 -10-5 M) xx = 7.73943 -10-6 MPlug in value of x to solve for KK = x/E xD xK = (7.739 -10-6 M)( 1.0 -10-5 M) (7.739 -10-6 M)( 1.0 -10-4 M) (7.739 -10-6 M)K = (7.73943 -10-6 M) 2.26057 -10-6 M9.22606 -10-5 MK = (7.73943 -10-6 M) (2.0856148 -10-10 M2)K = 37108.63 M-1K = 3.71 -104 M-1FINAL EXCEL WORKSHEET After refinementDISCUSSIONThe principle behind this lab experiment was to determine the binding co nstant of ethidium bromide to DNA using the known concentrations and the calculated value of X obtained from the equation. Ethidium bromide is said to have a high affinity to DNA, therefore, the expected value for the binding constant should be large. However, two methods were used to obtain the value K (binding constant). The first method was by using excel worksheet and inputting our data. The main goal was to manipulate the value in cell I24 to be as low as possible. After several trails and error, the final value of cell I24 was 0.00011316 and logK was 3.99, if you take the antilog of that value K would equal to approx. 9772.37, which is low in comparison to method two which was done by manual calculation. By manual calculation the obtained value for K was 37,108.63 M-1 which indicates that the final EB complex of DNA was larger than that of unbound DNA and unbound EB. The high value of the binding constant (K), demonstrates that there is a high affinity of ethidium bromide to D NA. This correlates to the expected values of EtBr, which are reported to be in between 104 M to 106 M.Using a buffer that does not contain added NaCl, such as BPE, will have unalike results than that of a buffer with NaCl, such as BPES. The BPE buffer will ante up a lower binding constant than that measured in BPES buffer. It is well known that the interaction within the process of intercalation is driven by electrostatic factors and -stacking with the bases (lab manual). The electrostatic binding of ethidium bromide to DNA has a preference to binding to the phosphate backbone the DNA strand. The DNA-ligand binding is salt-dependent due to the counter-ion release thats carried out during binding. This is indicative that the salt component in the buffer demonstrates a relatively greater stability in DNA due to its preference to the binding site within the GC-rich DNA region. With that said, it is apparent that an outlying positive charge is essential for intercalation. The positiv e charge on the intercalation diminishes as the aromatic system increases.Like ethidium bromide, Actinomycin D is another known intercalator with a high affinity to DNA. Though the two differ by means of binding sites. Actinomycin D intercalates at GC sites, which indicates that the two intercalators would not compete with one another at the exact binding sites. Therefore, by adding Actinomycin to a solution of herring testes DNA Ethidium bromide, resulting in two different results. An example of the plot is shown belowThe plot is simply an example of what it might look like. There are a lot of variables that must be considered in choosing the proper intercalator. Factors to consider are concentrations of solutions and DNA, buffers, whether its a low salt concentration or high salt concentration buffer. The difference in buffers could possibly yield two very different results. Another factor to consider is the magnitude of the absorbance. All these factors combined could hinder the final outcome, so it is hard to conclude exactly how the actinomycin D would react in combination with ethidium bromide to DNA within this experiment.REFERENCESJeremy M. Berg, J. L. (2015). Biochemistry 8th ed. Kate Ahr Parker.Eva M. Talavera, Pablo Guerrero, Francisco Ocana, and Jose M. Alvarez-Pez, Photophysical and Direct Determination of Binding Constants of Ethidium Bromide Complexed to E. coli DNA, Appl. Spectrosc. 56, 362-369 (2002)Fuller, W., and M. J. Waring. 1964. A Molecular mildew for the interaction of ethidium bromide with deoxyribonucleic acid. Ber. Bunsen Ges. Phys. Chem. 68805-808.Qiao C, Bi S, Sun Y, Song D, Zhang H, Zhou W (2008) Study of interactions of anthraquinones with DNA using ethidium bromide as a fluorescence probe. Spectrochim Acta A 70 136-143Graves, D. E., C. L. Watkins, and L. W. Yielding. 1981. Ethidium bromide and its photoreactive analogues spectroscopic analysis of deoxyribonucleic acid binding properties. Biochemistry. 201887-1892. PubMedSuh D, Chaires J B (1995) Criteria for the mode of binding of DNA binding agents. Bioorg Mediclin Chem 3(6) 723-728