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Bio301 Biochemistry I Midterm Examination 1, October 27, 2004 Question 1 Draw the following oligopeptides in their predominant ionic forms at pH 7. (Help: chemical formulas are given in Table 1) a) Phe-Met-Arg b) Gln-Ile-His-Thr Question 2 Normal human hemoglobin has an isoelectric pot of 6.87. A mutant variety of hemoglobin, known as sickle-cell hemoglobin, has an isoelectric point of 7.09. The titration curve of hemoglobin idicates that, in this pH range, 13 groups change ionization states per unit change in pH. Calculate the difference in ionic charge between molecules of normal and sickle hemoglobin. Question 3 Ionic strength, I is defined as Where ci is the molar concentration of the ith ionic species and Zi is its ionic charge. At high ionic strengths the solubilities ofproteins as well as those of most other substances, decrease. This effect is known as salting out. You have given 1.0 M solutions of NaCl, (NH4) 2SO4 and K3PO4.In which of these solutions would you expect that a protein be most soluble? Least soluble? Question 4 Estimate the molecular mass of fibrinogen using the figure given below. Would your estimate be more accurate if you determine the molecular mas from its electrophoretic mobility on SDS-polyacrylamide gel. Discuss. Question 5. What would be the relative positioning of the following proteins after they had been subjected to isoelectric focusing. Protein pI Insulin 5.4 Cytochrome c 10.6 Histone 10.8 Myoglobin 7.0 Ribonuclease 7.8 anode............................................ .......................................................cathode (+electrode) (-electrode) Question 6. Treatment of a polypeptide by dithiothreitol yields two polypeptides that have the following amino acid sequences: 1. Ala-Phe-Cys-Met-Tyr-Cys-Leu-Trp-Cys-Asn 2. 2. Val-Cys-Trp-Val-Ile-Phe-Gly-Cys-Lys Chymotrypsin catalyzed hydrolysis of the intact polypeptide yields polypeptide fragments with the following amino acid compositions: 3. (Ala, Phe) 4. (Asn, Cys2, Met, Tyr) 5. (Cys, Gly, Lys) 6. (Cys2, Leu, Trp2, Val) 7. (Ile, Phe, Val) Draw and indicate the positions of the disulfide bonds in the original polypeptide. (note: chymotrpsin cleaves bulky amino acids i.e. Phe, Tyr,Trp) Question 7: What structural features are responsible for the observations that -keratin fibers can stretch to over twice their normal length, whereas silk is nearly inextensible? Question 8: Compare (discuss the advantages and disadvantages) of x-ray crystallography and NMR as methods of atomic structure determinations. Question 9: The production of a small acidic protein, hCG (human chorionic gonadotropin) during pregnancy is the basis of most pregnancy test kits. Describe an easy method for detecting this protein. Explain each step of this method. Question 10: Would you recommend to a friend of yours, to purify a double-stranded DNA using a buffer at pH 4? Why or why not. Explain.