Lab4. Equilibrium Constants of Base Binding to Form DNA

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Feb 20, 2024

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Exploring Equilibrium Constants in Biological Systems http://chemcollective.org/vlab/86 Introduction In the ‘30s and ‘40s, biologists knew that the agent for heredity was in thread-like chromosomes, made of the molecule DNA. They also knew that DNA was made of four bases, (A)denine, (G)uanine, (C)ytosine, and (T)hymine. The race was on to be first to determine how they fit together, and how they carried hereditary information. 1 In 1953, James Watson and Francis Crick unraveled the mystery, by showing that DNA had the double-helix structure shown as: One strand of DNA spirals up the double helix. The opposing strand spirals down. The two strands have mirrored sequences such that a C is paired with a G, and an A is paired with a T. Procedure Copy this to your web browser. And allow your web browser to use “Flash”. http://chemcollective.org/vlab/86 In this virtual lab, you will be provided with stock solutions of all four bases. These are labeled dAMP for A, dCMP for C, dGMP for G, and dTMP for T to reflect the fact that these bases are only soluble as single bases in their d-MonoPhosphate form. Each of these solutions has a concentration of 0.10M. 1. Click Introductory Video and Support Information on the upper right corner to learn how to use this virtual lab. 2. Take a bottle of dAMP solution (0.1 M), a bottle of dTMP (0.1M), one 250 mL beaker, and two 25 mL pipettes on the work bench. 3. Withdraw 25 mL of dAMP solution into one of the pipette and deliver it into the beaker. 4. Withdraw 25 mL of dTMP solution into another pipette and deliver it into the same beaker. For this virtual lab, it will be perfectly fine if you use the same pipette to deliver both dAMP and dTMP solutions. However, to better resemble a real lab to avoid cross contamination, you should use two separate pipettes for the two different solutions. 5. Click on the beaker containing both dAMP and dTMP solutions and read the information window. Record the equilibrium concentrations of A, T and AT in Table 1 on the Data Sheet, Table 1. Also record the temperature of the solution. In this window, AT indicates A and T bound together. 1 Figure from Eric S. Grace, “Biotechnology Unzipped: Promises and Realities”, Joseph Henry Press, Washington, DC (1997). 1 From Wikimedia Commons

6. Make calculations about binding of the bases to form the base pair. The binding reaction equation is A + T  AT The equilibrium constant for this reaction is referred to as the “binding constant” for A and T, and ∆G o for the reaction is the “free energy of binding”. Determine the equilibrium constant and the free energy of the binding reaction. And show the calculation work in Data Sheet. 7. Design an experiment to determine the equilibrium constant and the free energy of binding reaction of C and G to form CG. Hint: repeat Step 1-6, except replace A with C, and T with G. Record the equilibrium concentrations of C, G and CG in the Data Sheet, Table 2. Determine the equilibrium constant and the free energy of the binding reaction between C and G. And show the calculation work in Data Sheet. 8. Design an experiment to determine the equilibrium constant and the free energy of binding reaction of A and C to form a mis-match pair AC. Hint: repeat Step 1-6, except replace C with T. Record the equilibrium concentrations of A, C and AC in the Data Sheet, Table 2. Determine the equilibrium constant and the free energy of the binding reaction between A and C. And show the calculation work in Data Sheet. Data Sheet Table 1: AT binding. The chemical equation is A + T  AT [A] eq [T] eq [AT] eq Temperature in o C Temperature in K K(binding of AT) = [ AT ] eq [ A ] eq [ T ] eq = _____________________________  G o (binding of AT) = -RT ln K= _____________________________ R is the gas constant 8.31 J/mol.K; T is the temperature in K. Table 2: CG binding The chemical equation is C + G  CG [C] eq [G] eq [CG] eq Temperature in o C Temperature in K K(binding of CG) = [ CG ] eq [ C ] eq [ G ] eq = _____________________________  G o (binding of CG) = -RT ln K = _____________________________ R is the gas constant 8.31 J/mol.K; T is the temperature in K. Table 3: AC mis-matched binding The mis-matching reaction is A + C  AC 2

[A] eq [C] eq [AC] eq Temperature in o C Temperature in K K(binding of AC) = [ AC ] eq [ A ] eq [ C ] eq = _____________________________  G o (binding of AC) = -RT ln K = _____________________________ R is the gas constant 8.31 J/mol.K; T is the temperature in K. Post Lab Assignments 1. Does your resultant K and  G o make sense in terms of the structure of DNA? In the DNA double helix, a C is paired with a G, and an A is paired with a T. Briefly explain. 2. Calculate the equilibrium constant at 25  C for the following reaction from the value of Δ G  given. Show your work. 3. Calculate Δ G  for the following reaction from the equilibrium constant at the temperature given. Show your work. 2SO 2 (g) + O 2 (g)  2SO 3 (g) T=500 O C K P =48.2 3

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4. To a new word file, paste the completed section of Data Sheet. Upload the file to Canvas when you take the online quiz. (5 points) 5. Take the online quiz (10 points). The quiz is based on Post Lab Assignments. Make sure you know how to answer the post lab questions before you take the quiz. The post lab questions themselves won’t be graded, instead, they are used for you to study for the quiz. 4

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