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Biochemistry I PS2 Recitation September 1, 2016 1. Go to the following URL and perform a pH titration. a) Collect data for the pH versus mls of NaOH added. Use Excel to plot your titration data. b) Determine the pKa and the type of acid (based on the list of pKa on the web page). https://andrew.cmu.edu/user/rule/bc_oli/Pset/PS02/titration.html 2. You are required to make a 250 ml solution of 0.05 M bis-tris to use as R [ A ] /[HA] 10 ( pH pKa) a buffer to perform a biochemical experiment at pH 6.0. You only have f HA 1 /(1 R) [ HA] /[ AT ] the fully deprotonated form of this acid in the lab, plus 1M solutions of HCl and NaOH. What additional information do you need to complete f A R /(1 R) [ A ] /[ AT ] this problem? a) Sketch the titration curve for this buffer on the right. b) How many moles of the deprotonated bis-tris do you need to add to make your buffer solution? c) Does your answer to b) depend on the pH of the solution? c) Calculate fHA and fA. Do your calculated values agree with what you would estimate from the titration curve? d) How many equivalents of HCl or NaOH do you have to add (specify what would be added to the solution)? e) How many moles of HCl or NaOH would you have to add? √ Have someone check your work 3. The protein shown on the right contains one histidine residue and one aspartic acid. a) What would you predict for the pKa of the histidine residue? Higher or lower than the normal pKa of 6.0? Why? [Hint: how will the negative charge affect the acidity of the histidine?] O O H N + N H N N H 100 b) Assuming the protein is only biologically active when the histidine is deprotonated, sketch the activity as a function of pH. [Hint: Is the activity proportional to fHA or fA- ?]. What is the x-axis of this plot? % activity 50 0 √ Have someone check your work. O O + + H You will use graphical methods to determine the net charge on the peptide (you could use the formula for fHA to get more accurate numbers). The overall charge (averaged over all the molecules) is just the weighted sum of the charges on each group: n i i qTOTAL ( f HA q HA f Ai q iA ) i 1 Fraction Protonated Biochemistry I PS2 Recitation 4. A tripeptide (Ala-Ala-His) is shown to the right, fully protonated at low pH (left) and pKa=6 fully deprotonated at high pH (right). This diagram indicates the charge on each ionizable group when protonated and deprotonated. The sidechain of histidine is pKa=9 circled in the right diagram. 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 September 1, 2016 pKa=2 a) Sketch the fraction protonated versus 0 1 2 3 4 5 6 7 8 9 10 11 12 pH for each group (amino, His pH sidechain, carboxyl) on the graph to the right. b) Calculate the charge at pH = 6. Indicate the contribution of each ionizable group to the overall charge on the diagram below. What are the predominate species in solution at this pH? √ Have someone check your work. 5. Sketch the titration curve for the amino acid glutamic acid, whose structure is shown below. a) Label the axes first, b) add the appropriate scale, in equivalents, on the bottom. (One equivalent of NaOH is equal to the number of moles of the weak acid). c) Indicate the buffer regions. pKa=4 pKa=2 pKa=9.0 √ Have someone check your work. 6. View the Jmol page that shows methane and butane dissolved in water. a) What do the dotted purple lines represent? b) Which of these two compounds would you expect to be less soluble in water? Why? c) Which of the two compounds would cause a larger decrease in the entropy of water? Why? √ Have someone check your work.