Name: Student Number: THE UNIVERSITY OF MANITOBA April 16, 2007, 9:00 AM -12:00 PM Page 1 (of 4) Biochemistry II Laboratory Section Final Examination MBIO / CHEM.2370 Examiner: Dr. A. Scoot 1. Answer ALL questions in the space provided. 2. The back side of each page may be used for your answer or for preliminary work. 3. Questions to invigilators about the exam will not be answered. 3 1. State which chemicals or products were used in the lab this term for each of the following purposes. N.B. Full names and correct spelling required. A) To act as the oxidized coenzyme in the staining sequence for lactate dehydrogenase. B) To form the polar phase for adsorption chromatography. C) To act as the substrate in the assay used to measure lysozyme activity. D) To act as an oxidizing agent in the assay used to measure blood glucose concentrations. E) To form a coloured complex in the assay used to measure blood glucose concentrations. F) To precipitate protein and DNA in order to separate them from RNA. 4 2. Name the assay used in the lab this term to measure RNA concentration and using a chemical equation describe the reaction that occurs in this assay. Make sure you include all reactants and conditions required for the reaction.
April 16, 2007, 9:00 AM -12:00 PM Page 2 (of 4) Biochemistry II Laboratory Section Final Examination MBIO / CHEM.2370 Examiner: Dr. A. Scoot 3. A tissue extract containing six proteins, PK, PL, PM, PN, PO and PP, was dissolved in 0.15 M phosphate buffer, ph 5.5. The molecular weights and isoelectric points of the proteins are shown below: Protein MW pi PK 107,500 10.2 PL 35,000 4.8 PM 173,000 7.3 PN 96,000 6.2 PO 185,000 8.6 PP 71,000 8.6 5 a) The tissue extract was placed on a CM-Sephadex column and subsequently eluted with three buffers to give three fractions. Fraction 1A eluted at ph 5.5; Fraction 2A eluted at ph 6.5; Fraction 3A eluted at ph 9.0. (i) Indicate which if any of the six proteins would be found in these three fractions. (N.B. Any protein will not be found in more than one fraction from a particular column) Fraction 1A Fraction 2A Fraction 3A (ii) Give reasons to explain the answer given above including a brief description of how the column functions and why the proteins behave as they do on the column.
April 16, 2007, 9:00 AM -12:00 PM Page 3 (of 4) Biochemistry II Laboratory Section Final Examination MBIO / CHEM.2370 Examiner: Dr. A. Scoot 4 b) Fraction 3A from the first column was then applied to a Sephadex G-100 column (Fractionation range MW 4,000-150,000) and eluted with buffer, ph 9.0, to give twelve fractions. Fractions 1B to 12B. Only two of these fractions contained protein, 1B and 6B. (i) Indicate which if any of the six proteins would be found in these fractions. Fraction 1B Fraction 6B (ii) Give reasons to explain the answer given above including a brief description of how the column functions and why the proteins behave as they do on the column. 2 c) Fraction 1B was then subjected to cellulose acetate electrophoresis at ph 8.6. Draw a carefully labeled diagram to show the results you would expect to see.
April 16, 2007, 9:00 AM -12:00 PM Page 4 (of 4) Biochemistry II Laboratory Section Final Examination MBIO / CHEM.2370 Examiner: Dr. A. Scoot 3 4. You are given a preparation containing an enzyme that converts X into Y. State what assays you would do (details not required) to determine the specific activity of this enzyme and indicate how specific activity would be calculated. Why is it useful to know the specific activity of a preparation? 4 5. A liver homogenate was prepared to look at the level of LDH. Under standard assay conditions 0.1 ml of a 1 in 20 dilution of the homogenate was found to calalyse the conversion of lactate to pyruvate at a rate of 7.5 moles pyruvate produced per min and 0.5 ml of a 1 in 100 dilution of the homogenate was found to contain 2.5 mg protein. A 10 ml sample of the homogenate was applied to a chromatography column and 5 ml fractions collected. The enzyme activity was found in a single fraction where 0.1 ml of a 1 in 60 dilution of the fraction catalysed the reaction at a rate of 3.0 moles pyruvate produced per min and 0.2 ml of a 1 in 10 dilution of the fraction was found to contain 4.0 mg protein. Calculate the degree of purification achieved by fractionation. Show ALL your calculations.
Name: Student Number: THE UNIVERSITY OF MANITOBA April 16, 2007, 9:00 AM -12:00 PM Page 1 (of 4) Biochemistry II Lecture Section Final Examination MBIO / CHEM 2370 Examiner: J. Stetefeld, P. Loewen 1. Answer ALL questions in the space provided. 2. The back side of each page may be used for your answer or for preliminary work. 3. Questions to invigilators about the exam will not be answered. 10 1. Using structural formulae, compound names and enzyme names, describe how pyruvate is incorporated into glycogen and how one glucose unit in glycogen is converted to glucose-6-phosphate. 7 2. Describe using diagrams, the mechanism by which protein kinase and protein phosphatase control glycogen metabolism.
April 16, 2007, 9:00 AM -12:00 PM Page 2 (of 4) Biochemistry II Lecture Section Final Examination MBIO / CHEM 2370 Examiner: J. Stetefeld, P. Loewen 10 3. Using diagrams, describe the processes in the ribosome, including all necessary protein factors and enzymes, by which a peptide bond is formed between the first methionine in the protein and a serine leaving the dipeptide ready for addition of a third amino acid. How much energy in ATP equivalents is required? 10 4. Using structural formulae, compound names and enzyme names describe how a molecule of palmitic acid is synthesized using threonine as the carbon source.
THE UNIVERSITY OF MANITOBA April 16, 2007, 9:00 AM -12:00 PM Page 3 (of 4) Biochemistry II Lecture Section Final Examination MBIO / CHEM 2370 Examiner: J. Stetefeld, P. Loewen 18 5. Using compound structures and names and enzyme names or diagrams where necessary, describe: a) the role of sigma factor ( ) in transcription; b) the role of CAP (catabolite gene activator protein) in transcription of the lac operon; c) the reaction catalyzed by nitrogenase complex and its role; d) the reactions of the glyoxalate shunt and their role; e) how light absorption on the chloroplast membrane supports a quantum yield of 8; f) the biochemical basis of phenylketonuria.
April 16, 2007, 9:00 AM -12:00 PM Page 4 (of 4) Biochemistry II Lecture Section Final Examination MBIO / CHEM 2370 Examiner: J. Stetefeld, P. Loewen 10 6. Using structural formulae and names of all intermediates and the names of enzymes and coenzymes, outline the steps required to convert CO 2, aspartate and ribose-5-phosphate into UMP. Assume an abundant supply of -ketoglutarate, ATP and coenzymes. An excess of aspartate also serves as the source of nitrogen. 10 7. Using structural formulae of all intermediates and the names of enzymes and coenzymes, describe the reactions that fix carbon dioxide into glyceraldehyde-3-phosphate in a C4 plant. How does this differ from the process in a C3 plant and what advantage does this provide to a C4 plant?