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Download FST 123 - Enzymology Homework IS `13
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FST 123 - Enzymology Homework I S '13 Name____________ 1. Briefly define the following terms: a. Hydrophobic effect b. Quaternary structure: c. Domain d. Primary structure e. Ramachandran diagram f. Salt Bridge g. Torsion angle h. Tertiary structure 2. If a protein is composed of 225 amino acids: a. What is its approximate molecular weight? What is its approximate volume? b. Assuming it is a sphere, what is its radius? 3. The course website contains a link to a Kinemage file depicting the structures of four proteins. Download them, view them using Mage or King (http://kinemage.biochem.duke.edu/software/index.php), and classify them according to Chothia’s four categories. 4. A buffer was made by dissolving 18.92 g of lactic acid and (pKa = 3.86) 32.71 grams of sodium lactate in 1L or water. (Mr.: Lactic acid, 90.08; NaLactate, 112.8). a. What is the pH of this buffer? b. What is the concentration of the buffer? c. What is the ionic strength of the buffer? 5. Protein A contains 30 carboxyl groups and 18 amino groups; protein B contains 15 carboxyl groups and 20 amino groups. Assume that the carboxyl groups have pKa's of 4.0 and that the amino groups have pKa's of 9.0. a. Neglecting second-order effects, estimate the pI of each protein. b. Which of the two proteins would be more likely to adsorb to a DEAE-cellulose chromatography column in buffer of low ionic strength at pH 8.2? What about a CMcellulose column at pH 6.2? c. From the following table, select the acids that would be the best choices for making the two buffers described in part b. acid formic acetic benzoic Ka acid carbonic 4.3 x 10-4 1 1.7 x 10-5 MES 2 6.4 x 10-5 Phosphoric 1MES = 2-[N-morpholino]ethanesulfonic acid 2the second ionization is given for phosphoric acid. 3Tris-Cl = tris(hydroxymethyl)aminomethane•HCl. Ka 4.3 x 10-7 6.3 x 10-7 6.2 x 10-8 acid hypochlorous Tris-Cl3 hypoiodous Ka 3.0 x 10-8 7.1 x 10-9 2.3 x 10-11 6. The following information is known about four proteins: Protein A B C D pI 5.2 9.1 6.2 5.8 Monomer Mr 13,100 14,200 66,700 50,000 subunits 1 1 2 identical, 66.7 k each 4 identical, 50,000 each a. Sketch below, the elution profile expected if this mixture is run on a Sephadex G-100 gel filtration column run in 50 mM phosphate buffer, pH 6. Label the peaks. b. On the following page, sketch the gel you would expect to obtain from running these proteins on an SDS polyacrylamide electrophoresis gel at pH 8.0, (after staining and destaining). c. What predictions can you make about the results of a native PAGE at pH 7.6 (State any assumptions you might need to make about the % acrylamide in the gel.) d. Sketch the elution profile of these proteins from a carboxymethyl cellulose ion exchange chromatography column, run at pH 6.25 (with a salt gradient, if necessary). Label the peaks. G-100 Column SDS Gel CMC column 2 7. List two distinctly different criteria used to assess the purity of an enzyme and briefly discuss their relative merits. Note that sometimes you think a protein is pure, but you have really just been unable to separate the impurities, so you don’t know they are there. It’s always a good idea to use more than one technique. 8. Complete the following purification table: Step Crude extr total vol (mL) 500 Protein (mg/mL) 2.0 Activity (U/mL) total protein (mg) total activity sp Act (U/mg) Purification (fold) Recovery (%) 6.2 20% Ammon. 18 6.7 4 sulfate ppt 20% Ammon. 499 1.6 5 sulfate super. 40% Ammon. 10 2.1 169 sulfate ppt 40% Ammon. 550 1.5 1.4 sulfate super. (40% ammonium sulfate super. means the supernatant solution obtained from the 20% ammonium sulfate supernatant by adding enough ammonium sulfate to bring the concentration to 40% of saturation, then centrifuging it.) 3