Chem153A Spring 2013 Midterm #1 Form C Last Name:_____________________________ Page 1 of 7 Chem 153A Spring 2013 Midterm #1 Form C April 22, 2013 _____ ____ ___ ___ _ Name (Last) (First) Student ID Lecture Page Score Be sure to read all the questions carefully, and answer the question that is asked. Answer questions as concisely as possible. The word limits are more than sufficient to fully answer the question. Nothing over the word limit will be graded. 2 (19 pts) 3 (20 pts) 4 (17 pts) 5 (18 pts) 6 (14 pts) 7 (12 pts) Total (100pts):Chem153A Spring 2013 Midterm #1 Form C Last Name:_____________________________ Page 2 of 7 pKa’s you may need for this page: NH3=8.0 (in a peptide) NH3=9.5 (free amino acid), COOH = 3.5 (in a peptide), COOH = 2 (free amino acid), R-groups: ASP = 3.9, GLU = 4, HIS = 6.0, LYS = 10.5, ARG = 12.5 1. a.(7 pts) Draw the following peptide at physiological pH: C – L – E – A – R b. (1 pt) What is the net charge on the peptide? 0 c. (3 pts) What is the pI of the peptide?(Show your work) pI = (4.0 + 8.0)/2 = 6.0 d. (1 pt) How many peptides could be made with the same LENGTH? 205 = 3,200,000 e. (7 pts) Sketch the titration curve for the above peptide on the graph below. Indicate on the graph where it would act as a good buffer. 012340 1 2 3 4 5 6 7 8 9 10 11 12 13 14OH- equivalents pHChem153A Spring 2013 Midterm #1 Form C Last Name:_____________________________ Page 3 of 7 2. a. (7 pts) You want to make a Phosphate buffer, containing a total of 0.6 M weak acid and conjugate base, at pH 6.2. On the chemical shelf you have H3PO4, KH2PO4, K2HPO4, and K3PO4. (pKas are 2.1, 6.9, and 12.32). Which chemicals will you use and how much of each should you add to 1 liter of water. (Show all your work) KH2PO4  K2HPO4 pKa = 6.9 6.2 = 6.9 + log (A-/HA) A-/HA = 0.2 A- = HPO42- = 0.2/1.2 x 0.6M = 0.1M HA= H2PO4- = 1.0/1.2 x 0.6M = 0.5M b. (5 pts) You also need to make a buffer at pH 8.5. Could any of these chemicals be used to make a good buffer at pH 8.5? Why or Why not? (30 words maximum) No, Since no pKas are within 1 pH unit of 8.5 there would not be sufficient weak acid or conjugate base to resist pH changes with the addition of acid or base. 3. (8 pts) The sequence shown below forms an alpha helix in the yeast protein Erv1. G – S – R – T – Y – R – K – V – D – P – P – D – V – E – Q – L – G – R – S – S – W a. List ALL the interactions in this sequence that would stabilize this structure. - Hydrogen Bonds between backbone atoms - Arg-Asp - Glu-Arg b. You would NOT predict this sequence could form an alpha-helix. List the interactions that you would expect to destabilize the alpha-helix. - Prolines - Arg-Arg - Arg-Lys - Asp-AspChem153A Spring 2013 Midterm #1 Form C Last Name:_____________________________ Page 4 of 7 4. As a graduating Ph.D. student, for the past 5 years you have been studying an enzyme that catalyzes the oxidation of isocitrate (an intermediate in the citric acid cycle). You have experimentally determined that the pI of the protein is 4.8 and that it is soluble until 3.7M Ammonium Sulfate at that pH. a. (7 pts) With this information what techniques could you use to purify this protein? Describe the steps you would take for one of them. Include any special conditions you would need to consider with that technique. - Salting Out (or Ammonium sulfate fractionation) o Set the buffer pH to the pI of the protein o Add salt to below 3.7 M and remove precipitated proteins o Add salt to over 3.7 M and isolate protein of interest. - Ion exchange chromatography o Use a cation exchange column and set the buffer pH <4.8 OR Use an anion exchange column and set the buffer pH > 4.8 o Load protein (from salting out) onto column, Protein of interest would bind to the column o Elute protein off column with change in salt concentration OR change in pH Explanation of Only One Technique Graded b. (4 pts) List two things (there are many) that you would want to confirm or determine after you have purified your protein, but before conducting experiments, and what technique you would use for each. Protein Amount: Protein assay (dye binding, or absorbance spectroscopy) Protein is Pure: SDS-PAGE Protein is folded: circular dichroism Protein is functional: Activity Assay 5. (6 pts) Describe the roles of polysaccharides in biological systems. Include structural differences that make different polysaccharides uniquely suited for each role. (50 words Maximum) Structural (cellulose) and energy storage (glycogen or starch) Structural polysaccharides have Beta glycosidic linkages which increase strength of the molecule because they are indigestible by most organisms Energy storage polysaccharides are highly branched with alpha glycosidic linkages, allowing for quick mobilizationChem153A Spring 2013 Midterm #1 Form C Last Name:_____________________________ Page 5 of 7 6. a. (4 pts) Describe the structural similarities and differences between Myoglobin and Hemoglobin. (30 words maximum) Similarities: the polypeptides adopt the globin fold and bind Heme as a prosthetic group to bind oxygen Differences: Mb is a monomer while Hb is a tetramer b.(4 pts) How does Hemoglobin’s structure make it better suited for its function? Since Hb is a tetramer is can exhibit cooperativity (i.e. switch between affinity states) and it forms a central cavity that 2,3-bisphosphoglycerate can bind to This allows Hb to efficiently transport oxygen from the lungs to the tissues c. (8 pts) How would an increase in muscle activity affect the function of Hemoglobin? Include the molecules that would directly contribute to this affect and draw oxygen binding curves to illustrate your answer. (30 words maximum) An increase in muscle activity would produce CO2 and protons (lowering the pH near the tissues) both of these are negative regulators of Hb and thus would decrease oxygen binding in the tissues. d. (2 pts) Why would this situation be beneficial? (30 words maximum) This ensures that more oxygen is delivered to actively respiring tissues, which would require more oxygen. Normal Activity Increased activityChem153A Spring 2013 Midterm #1 Form C Last Name:_____________________________ Page 6