BCH 4053 PRE-TEST 1 GROUP NAME _____________________ May 29, 1996 This test is take-home and open book, and it is intended that all members of the group contribute to completing it. Only one copy is to be submitted by the group, and all members who participated should sign their names below. Test is due by 1:30 pm on Monday, June 3. __________________________ __________________________ __________________________ __________________________ __________________________ __________________________ Page Points 1 _____ 2 _____ 3 _____ 4 _____ 5 _____ 6 _____ Total _____ Points (12) 1. Draw a titration curve for the dipeptide Val.His on the graph below. Locate and identify the points on the curve corresponding to pK1, pK2, and pK3. Calculate the approximate pI value and locate its position on the curve. Indicate the pH region on the graph in which at least 25% of the histidine side chain is charged. ž 13 Ćž ³ pI = 12 Ćž ³ 11 Ćž ³ 10 Ćž ³ 9 Ćž ³ 8 Ćž ³ 7 Ćž pH ³ 6 Ćž ³ 5 Ćž ³ 4 Ćž ³ 3 Ćž ³ 2 Ćž ³ 1 ĆÄÄÄÄÄÄÄÄÄÄÄÄÄÄĮÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄĮÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄĮž 1 2 3 equivalents of base -----> (5) 2. Acetoacetic acid, a ketone body produced by the liver and used as a fuel by other tissues, has a pK of 3.58. What fraction of the acetoacetic acid molecules in urine having a pH of 4.48 would be in the protonated, uncharged form? (Note: I'm looking for fraction, not ratio). (10) 3. You have solutions of (a) 1.0 M NaOH and (b) 1.0 M H3PO4, and you would like to prepare 2 L of 0.05 M phosphate buffer at pH 7.0. Calculate the volume of solutions (a) and (b) and water that must be combined to produce the buffer. (Assume volumes are additive.) For H3PO4: pK1 = 2.12; pK2 = 7.21; pK3 = 12.67 (10) 4. Given the following data on three different proteins: Protein: hemoglobin chymotrypsinogen urease Molecular Weight (M) 64,500 23,250 482,000 Diffusion Coefficient (D) 6.9 9.5 3.5 Isoelectric pH (pI) 6.8 9.5 5.0 Indicate in the blanks which of the three proteins will: ____________________ (a) Elute first from a gel filtration column. ____________________ (b) Elute first from a diethylaminoethyl cellulose ion exchange column. ____________________ (c) Have the smallest frictional coefficient (f). ____________________ (d) Migrate fastest upon electrophoresis in sodium dodecyl sulfate (SDS). ____________________ (e) Migrate fastest to the anode in a native electrophoresis experiment at pH 6.0. (18) 5. Underline the following peptides which are negatively charged at pH 7.0. Circle each amino acid which is non-polar. gln.val.tyr.ala lys.arg.glu.trp met.his.leu.asp cys.pro.gly.asn F-A-D-S M-I-C-K (6) 6. Describe two methods of cleaving disulfide bonds in proteins prior to sequencing. 7. Consider the following peptide: AEMKHS (5) (a) Calculate the pI. (You may either use actual pK values from page 113, or the approximate values you were asked to learn. On the regular test you would use approximate values.) Sketch enough of the structure to show how you arrive at your calculation. (6) (b) Treatment with cyanogen bromide produces two peptides. You want to separate these peptides from starting material and from each other. Predict the order of elution of the two product and one starting peptide from a cation exchange column eluted with a buffer of increasing pH. Explain your reasoning. (4) (c) Draw the structure of the first two PTH-amino acids produced in the first two rounds of the Edman degradation of the above peptide. PTH- amino acids are analyzed by reversed phase HPLC, similar to the analysis of PTC amino acids (Fig. 4-20, p. 4-23), in which the derivatives are eluted in order of decreasing polarity (most polar first). Predict the order of elution of these two PTH-amino acids from the HPLC column. (10) 8. The conformation of a peptide bond can be described by plotting a Ramachandran map, in which the ž and č angles are plotted against each other. (a) The ž angle represents rotation about which bond? (b) The č angle represents rotation about which bond? (c) Draw a Ramachandran map below, showing the approximate locations of the following conformations: alpha helix (right handed), į-sheet (parallel), collagen helix (6) 9. The fraction of ligand binding sites (ž) on myoglobin and hemoglobin can be expressed by the relationship ž = (pO2)n/[(pO2)n + (P50)n] where pO2 is the pressure of oxygen, P50 is the oxygen pressure required to occupy 50% of the sites, and n is the Hill coefficient. For myoglobin, P50 = 1 torr and n = 1. For hemoglobin, P50 = 26 torr and n = 2.8. Calculate ž for the two proteins in tissues where pO2 = 30 torr. (8) 10. The following graph shows three idealized curves that describe oxygen binding to hemoglobin. Identify by letter the curve or curves which represent the situations described in the questions below. ž ³ ³ A ³ ³ B Y ³ ³ C ³ ³ ĄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄž pO2 (a) A comparison of fetal hemoglobin to normal hemoglobin A could be represented by comparing ______ to _______. (b) Increasing the concentration of BPG in the red cell will cause a shift in oxygen binding similar to the shift from ______ to _______. (c) Acclimation to high altitudes will cause a shift in oxygen binding similar to the shift from _______ to _______. (d) Increasing the pH of the hemoglobin solution from 7.2 to 7.4 will cause a shift in oxygen binding similar to the shift from _______to _______.