Student Number: A 10 ml volume of the skeletal muscle extract was applied to each of the two columns.

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1 Name: Student Number: THE UNIVERSITY OF MANITOBA April 21, 2010, 1:30 PM -4:30 PM Page 1 (of 4) Biochemistry II Laboratory Section Final Examination 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. 1. Two different chromatography columns were used to isolate lactate dehydrogenase from a skeletal muscle extract prepared in 0.1 M TRIS buffer, ph 8.0. The protein content of 1.0 ml of a 1 in 10 dilution of the extract was found to be 4.0 mg. Enzyme activity in 0.1 ml of a 1 in 100 dilution of the extract was found to be 2.0 enzyme units. One unit of enzyme activity is measured as the oxidation of one micromole of NADH per minute. A 10 ml volume of the skeletal muscle extract was applied to each of the two columns. With column 1 (Sephadex G-100, MW range ,000) the enzyme was eluted in fraction 1 (the first of 12 fractions) where the fraction volume was 5 ml. The protein content of the fraction was 4.0 mg/ml and the enzyme activity in 0.1 ml of a 1 in 100 dilution of the fraction was 2.0 enzyme units. With column 2 (CM-Sephadex, ph 8.0) the enzyme was eluted with 0.1 M TRIS, ph 8.0, in fraction 1 where the fraction volume was 20 ml. The protein content of the fraction was 4.0 mg/ml and the enzyme activity in 0.1 ml of a 1 in 40 dilution of the fraction was 2.0 enzyme units. 3 (a) Which of the two columns gave the best purification of the enzyme? You must show all calculations and cite purification values to support your answer.

2 April 21, 2010, 1:30 PM -4:30 PM Page 2 (of 4) Biochemistry II Laboratory Section Final Examination Examiner: Dr. A. Scoot 2 (b) What does the elution profile from each of the two columns tell you about the molecular weight of the enzyme? Use one sentence to briefly explain each of your answers. Column 1: Column 2: 2 (c) What does the elution profile from each of the two columns tell you about the pi of the enzyme? Use one sentence to briefly explain each of your answers. Column 1: Column 2: 1 (d) If the fractions containing the lactate dehydrogenase were subjected to electrophoresis at ph 8.8 which direction would the enzyme migrate on the cellulose acetate strip? Briefly explain your answer. 2 (e) When the extract was treated with urea and then applied to column 1 the enzyme activity was found in fraction 9 instead of fraction 1. Explain this observation.

3 April 21, 2010, 1:30 PM -4:30 PM Page 3 (of 4) Biochemistry II Laboratory Section Final Examination Examiner: Dr. A. Scoot 3 2. 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 visualise lactate dehydrogenase by changing from colourless to purple in the staining sequence. Ans B) To act as the oxidized coenzyme in the staining sequence for lactate dehydrogenase. C) To act as the moving solvent for the separation of plant pigments by adsorption chromatography. D) To act as the stationary phase for separation of plant pigments by paper chromatography. E) To yield a purple complex upon reaction with peptide bonds in the assay to measure protein concentration. H) To degrade RNA in order to be able to separate it from DNA and protein Very briefly, in a few lines, describe the basis of the enzyme assay you used in the lab this term for lysozyme.

4 April 21, 2010, 1:30 PM -4:30 PM Page 4 (of 4) Biochemistry II Laboratory Section Final Examination Examiner: Dr. A. Scoot 5 4. Briefly describe the chemical assay used in the lab this term to measure blood glucose concentrations in the glucose tolerance test. Your answer should indicate the reagents used and their purpose however specific concentrations and volumes of these reagents are not required Name the assay used in the lab this term to measure RNA concentration and using only an equation indicate the reaction that occurs in this assay. Make sure you include names of all the reactants and conditions required for the reaction, structures are not required.

5 Name: Student Number: THE UNIVERSITY OF MANITOBA April 21, 2010, 1:30 PM -4:30 PM Page 1 (of 4) Biochemistry II Lecture Section Final Examination Examiners: Drs. M. Spearman, 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 Using structural formulae and the names of compounds, enzymes and coenzymes, describe the steps required for the biosynthesis of isoleucine from butyryl-coa using glutamate as the amino donor.

6 April 21, 2010, 1:30 PM -4:30 PM Page 2 (of 4) Biochemistry II Lecture Section Final Examination Examiners: Drs. M. Spearman, P. Loewen Using structural formulae of all intermediates and the names of enzymes and coenzymes, describe the reactions that convert ribulose-5-p and CO 2 into two glyceraldehyde-3-p in a C4 plant. Indicate clearly how this differs from the process in a C3 plant including the amount of energy required Using diagrams and a short explanation, that include the names, locations and roles of all components, outline the process that produces ATP and NADPH for CO 2 fixation. What is meant by "quantum yield" in relation to photosynthesis?

7 April 21, 2010, 1:30 PM -4:30 PM Page 3 (of 4) Biochemistry II Lecture Section Final Examination Examiners: Drs. M. Spearman, P. Loewen Using structural formulae and the names of compounds, enzymes and coenzymes, describe the series of reactions required to convert an excess of threonine into palmitoleic acid in bacteria. Assume an abundant supply of all needed cofactors Using structural formulae and diagrams, outline the steps leading to the formation of the translation initiation complex starting with methionine. Assume an abundant source of all needed cofactors.

8 April 21, 2010, 1:30 PM -4:30 PM Page 4 (of 4) Biochemistry II Lecture Section Final Examination Examiners: Drs. M. Spearman, P. Loewen Using diagrams, structural formulae of all intermediates and the names of enzymes and coenzymes, describe the biosynthesis of dcdp using ribose-5-p as the source of the carbohydrate portion, and glutamine, CO 2 and oxaloacetate as the source of atoms in the pyrimidine ring Using diagrams, describe the role of σ(sigma) factor in transcription.