BIOL/CHEM 3361 Biochemistry I(and BIOL 6352)Spring 2016Due: Mon. Feb. 2 at 5:00 pm in the collectionbox in the hall outside FO 3.606(No late Problem Sets will be accepted.)(To void last minute difficulties you may turnthem in early — at lecture or dropped in thecollection box.)PROBLEM SET 1There are 31 lettered subparts to the questions below. Each will be graded on a 5 point scale and thenthe summed score will be converted to a percentage of the total points.For full credit all steps to the solutions of the following problems must be shown. You may worktogether on the problems but you may not copy or plagiarize. Your answers must be written mustshow your own math steps and must be in your own words.Round your answers as appropriate but in no case to no more than three significant figures.For problems 1 and 2 assume an activity coefficient of 1 for all substances and no effect of ionicstrength. Eliminate terms in quadratic solutions for [H ] only if the weak acid is dissociated < 5%.Reported pKa values can vary depending on the conditions under which they were measured; therefore in solving the following problems use the pKa values given with the problems.1. a. What mM concentration of HBr gives a pH of 1.3?What is the pH of 10 mM NaOH?At equilibrium 0.1 M nitrous acid (HNO2) produces 7.4 mM NO2-. Calculate the pKa of HNO2.b. What is the pH of 0.5 M benzoic acid? Benzoic acid Ka = 6.46 x 10-5 Mc. What is the pH of 45 mM H3PO4?For c-f: Phosphoric acid (H3PO4) is a triprotic acid; pKa1 = 2.12 pKa2 = 7.21 pKa3 = 12.32Acetic acid pKa = 4.75d. What is the ratio of H2PO4- to H3PO4 at a pH of 3?e. If 2 volumes of 18 mM KOH are mixed with 1 volume of 22.5 mM H3PO4 what will be the pHof the final mixture?f. If 15 µmoles of acetic acid is generated in a 1.0 ml enzymatic reaction buffered by 50 mM Naphosphate (pH 7.0) what will be the final pH of the reaction mixture?Would the change in pH be smaller or larger if the reaction were buffered by 50 mM Naphosphate (pH 6.0)? Explain you answer; no calculations required.2. a. Parietal cells control the concentration of HCl in the stomach by secreting H and Cl-. Whilefasting they maintain stomach pH at 3.0. After a meal they decrease stomach pH to 1.4. Whatis the fold-change in [H ] from fasting to after a meal?b. For every H ion secreted into the stomach parietal cells also secrete a HCO3- ion into bloodplasma. HCO3- plays an important role in maintaining the pH of blood plasma (see textbookpage 45 for equations). Patients with achlorhydria have impaired HCl secretion and thus have aloss of HCO3- in the blood. What will be the blood pH of a achlorhydria patient with half thenormal concentration of HCO3- in the blood? Assume a constant CO2(d) of 1.2 mM and anormal HCO3- of 24 mM. c. The pH of the stomach can influence the ionization of drugs such as Aspirin (aka acetylsalicylicacid or HAsp). Given that at equilibrium the pH of a 0.1 M Aspirin is 2.24 what is the pKa ofAspirin?d. Using the pKa from c. what is the ratio of Asp- to HAsp in the stomach of the patient after ameal?Would the ratio of Asp to HAsp increase decrease or stay the same if the patient had fasted?3. Two amino acids can join together via a peptide bond to make a dipeptide. Below is an example ofsuch a reaction.glutamate cysteine ?-glutamylcysteine?G°’ at 25°C for this reaction is 10 KJ/mol; assume 25°C in answering the following.a. Does the ?G°’ indicate that formation of ?-glutamylcysteine is favored? Explain your answer;no calculations required.b. What is Keq’ for this reaction?c. In certain conditions the formation of ?-glutamylcysteine can be coupled to ATP hydrolysis.ATP ADP Pi (?G°’=-30.5 kJ/mol).What is the overall ?G°’ for the coupled reaction? Is the formation of ?-glutamylcysteinefavored?d. What is Keq’ for the coupled reaction?e. If glutamate ATP ADP Pi are present at 20 mM 1 mM 0.2 mM and 3 mM what is theequilibrium ratio of ?-glutamylcysteine to cysteine?f. If ?-glutamylcysteine is removed such that the ratio decreases 10-fold and then 100-fold whatwill be the resulting change in ?G° (? ?G°)?4. Table 3.1 of your G&G text gives some thermodynamic values for the denaturation of severalproteins i.e. unfolding of the polypeptides under various conditions e.g. moderately high or lowpH. Among the proteins are myoglobin and RNase. Later in your text Fig. 6.30 shows thefractional unfolding of RNase A and B as a function of temperature.Figure 6-30 p168 a. Use the temperatures in Fig. 6.30 at which RNase A is 25 50 and 75% unfolded to make a van’tHoff plot. Use Excel to tabulate your data and make the plot. Include a trendline and itsequation.b. From the plot determine H´ and S´ for the denaturation reaction. What are thecorresponding values for RNase A folding?c. What is ?G°´ for RNase A folding at 30? In making this calculation assume H´ and S´ areconstant with respect to temperature.d. Is the RNase folding driven by entropy or enthalpy? State the basis for your answer.e. How do the thermodynamic values you calculated compare to those for myoglobin and RNase inTable 3.1? What could explain any differences?5. Ser Thr and Tyr are well known targets for post-translational phosphorylation. A lesser knowntarget is His [Kee and Muir ACS Chem. Biol. 2012 7 (1) pp 44–51] which can bephosphorylated at either the N-1 or N-3 positions of the imidazole group. One hindrance instudying this phosphorylation is its chemical lability especially at low pH. Nevertheless Hisphosphorylation is now recognized as an important signaling mechanism in prokaryotes and lowereukaryotes and is being associated with mammalian cellular processes cancer and inflammation.In E. coli the RcsD protein is part of a phosphorelay signal transduction system that acceptsphosphate from RcsC becoming phosphorylated on the His residue in the target sequenceSDFAALAQTAHRLKGVFAMLN. Assume you have synthesized this sequence and achieved invitro phosphorylation of the His by transfer from RcsC. Chemical characterization of theoligopeptide reveals the following pKas:N- and C-terminal 9.5 and 4.5 respectively; Asp side chain 4.3; Lys side chain 10.1; Arg sidechain 12.0; and His side chain 5.5. Upon phosphorylation the His imidazole pKa shifts to 6.5; thephospho group has pKas of 2.0 and 6.8.a. Write the single letter sequences of the His- and pHis-oligopeptides and indicate above thesequence the charges the residues will carry at pH 7.0. Indicate partial charges (i.e. 10% 90% charged) with d and d-.b. Calculate the pI of each oligopeptide.c. If a mixture of the His- and pHis-oligopeptides were passed through a column containingDEAE-matrix at pH 7.0 how would they behave? Explain why.d. If a mixture of the two oligopeptides were reacted with cyanogen bromide followed by V8protease digestion could the His and pHis-containing peptides be separated by DEAEchromatography at pH 7.0? Explain why. 6. Assume you are given a mixture of proteins that you analyze by standard 2-D electrophoresis withthe following results for isoelectric points and apparent molecular weights: protein A (Mr 45 140;pI 7.52) protein B (Mr 74 400; pI 5.21) and protein C (Mr 108 370; pI 6.26).a. In the SDS-PAGE step in what order did the proteins band starting nearest the anode and goingtoward the cathode? Explain why.b. Which protein would be considered the most basic? Explain why.c. If you subject the mixture of proteins A B and C to gel filtration on a 100 ml Sephadex G200column and find that C elutes at 40 ml and A at 80 ml at what volume will protein B elute?d. If protein D (Mr 91 385; pI 6.66) were added to the mixture of proteins A B and C and the newmix subjected to SDS-PAGE at what position would D be found in the gel relative to proteinsB and C? Express you answer as the fractional distance between proteins C and B at which Dbands i.e. CD distance/CB distance.e. If the mixture of A B and C is subjected to salting out with ammonium sulfate at pH 6.5 inwhat order would the proteins precipitate? Explain why.f. If the mixture of A B and C is subjected to P-cellulose (pKa1 3 pKa2 6) chromatography whatoptimal pH range should you use for the buffer and in what order will the proteins emerge fromthe column? Explain why.
