Name: Student Number: April 14, 2001, 1:30 AM - 4:30 PM Page 1 (of 4) Biochemistry II Lab Section Final Examination Examiner: Dr. A. Scoot 1. Answer ALL questions in the space provided.. 2. The last page provides space for preliminary work. 8 1. State which chemicals or products were used in the laboratory this term for each of the following purposes. N.B. The chemicals or products must be given their full names and be spelled correctly. (a) To produce a purple precipitant band in the final step of the staining sequence for lactate dehydrogenase. (b) To form the polar phase when adsorption chromatography was used. (c) To act as the substrate in the assay used to measure lysozyme activity. (d) To act as an oxidising 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 bring about a change in the structure of RNA to facilitate its separation from DNA. (g) To form a coloured complex in the assay used to measure DNA concentrations. (h) To yield a purple complex upon reaction with peptide bonds.
April 14, 2001, 1:30 AM - 4:30 PM Page 2 (of 4) Biochemistry II Lab Section Final Examination Examiner: Dr. A. Scoot 2. The purification of lactate dehydrogenase from a skeletal muscle extract prepared in 0.1M TRIS buffer, ph 8.0 was compared using three different columns. The protein content of 1.0 ml of a 1 in 10 dilution of the extract was found by biuret assay to be 4.0 mg. The 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 min. A 10 ml aliquot of muscle extract was applied to each of the three columns. With column 1 (Sephadex G - 100, MW range 4000-120,000) most of the enzyme appeared in fraction 1 (the first of twelve fractions) where the fraction volume was 5 ml. The protein content of the fraction was 4.0 mg/ml while the enzyme activity in 0.1 ml of a 1 in 100 dilution of the fraction was 2.0 enzyme units. With column 2 (Sephadex G - 150, MW range 5,000 to 300,000) the fraction containing most of the enzyme was number 10 (the tenth of twenty fractions) where the fraction volume was 10 ml. The protein content of the fraction was 5.0 mg/ml and the enzyme activity in 0.1 ml of a 1 in 40 dilution of the fraction was 2.0 enzyme units. With column 3 (CM-Sephadex ph 8.0) the enzyme was eluted with 0.1M TRIS, ph 8.0 in fraction 1 where the fraction volume was 20 ml. The protein content of the fraction was 4 mg/ml and the enzyme activity in 0.1 ml of a 1 in 40 dilution of the fraction was 2.0 enzyme units. For the following show all your calculations and where appropriate explain your answers. 4 (a) Which of the three columns would you choose if you were interested in the best percentage yield of enzyme?
April 14, 2001, 1:30 AM - 4:30 PM Page 3 (of 4) Biochemistry II Lab Section Final Examination Examiner: Dr. A. Scoot 5 (b) Which of the three columns gave the best purification of the enzyme? 2 (c) What does the elution profile from each of the three columns tell you about the molecular weight of the enzyme? Column 1 Column 2 Column 3
April 14, 2001, 1:30 AM - 4:30 PM Page 4 (of 4) Biochemistry II Lab Section Final Examination Examiner: Dr. A. Scoot 2 (d) What does the elution profile from each of the three columns tell you about the pi of the enzyme? Column 1 Column 2 Column 3 2 (e) If the fractions containing the lactate dehydrogenase were subjected to electrophoresis at ph 8.0 which direction would the enzyme migrate on the cellulose acetate strip? Explain your answer. 2 (f) If the extract was treated with urea and then applied to column 1 the enzyme activity would appear in fraction 9. Explain this observation.
April 14, 2001, 1:30 AM - 4:30 PM Working Page Biochemistry II Lab Section Final Examination Examiner: Dr. A. Scoot Working Space - (Not to be Marked)
Name: Student Number: April 14, 2001, 1:30 AM - 4:30 PM Page 1 (of 7) 1. Answer ALL questions in space provided. 2. The last page provides space for preliminary work. 10 1. Using structural formulae and names for all intermediates, and the names of enzymes and coenzymes, describe the pathway by which pyruvate is converted into squalene. Indicate which atoms in the intermediate isopentenyl pyrophosphate (either 2 or 3 ) would be radioactively labelled if the pyruvate was labelled with 14 C in the methyl group (C3).
April 14, 2001, 1:30 AM - 4:30 PM Page 2 (of 7) 12 2. Glucose-6-phosphate, labelled at position 2 with 14 C, is added to a solution containing all the components necessary for operation of the pentose phosphate pathway. Using structural formulae and names for all intermediates, and the names of enzymes and coenzymes, describe how ribulose-5-phosphate is formed and how it is converted into fructose-6-phosphate in two separate reactions of the pathway. Indicate clearly the pattern of 14 C-labelling in the two fructose-6-phosphate molecules. What would be the fate of the reduced electron carrier produced in these reactions in fat cells?
April 14, 2001, 1:30 AM - 4:30 PM Page 3 (of 7) 12 3. Using structural formulae and names of all intermediates, and the names of enzymes and coenzymes, describe how aerobic animal tissue might synthesize glycogen using aspartate as the sole carbon source. Assume an unlimited supply of aspartate, enzymes, and cofactors.
April 14, 2001, 1:30 AM - 4:30 PM Page 4 (of 7) 10 4. Using structural formulae and names for all intermediates, and the names of enzymes and coenzymes, describe how an atom of nitrogen, originally in nitrogen gas N 2, is converted into the amino group of aspartate. Assume pyruvate is the carbon and energy source.
April 14, 2001, 1:30 AM - 4:30 PM Page 5 (of 7) 10 5. Using a diagram, describe the Z-scheme of the light reactions of photosynthesis. Also using structural formulae and names for all intermediates, and the names of enzymes and coenzymes, describe the relevant energy utilizing steps (four in total) of the dark reactions. From this information, deduce the number of photons used, the number of NADPH, ATP and oxygen atoms generated and/or used during the photosynthetic fixation of one CO 2 with ribulose-5-phosphate to produce two molecules of glyceraldehyde-3-phosphate.
April 14, 2001, 1:30 AM - 4:30 PM Page 6 (of 7) 11 6. Given all components necessary for protein synthesis, use diagrams to describe how the first peptide bond to produce formylmethionyl serine is formed starting with methionine and serine. Be sure to draw the structure of the product. Indicate how many ATP equivalents are used in this process and justify your answer.
April 14, 2001, 1:30 AM - 4:30 PM Page 7 (of 7) 10 7. Using structural formulae and names for all intermediates, and the names of enzymes and coenzymes, describe the pathway by which a bacterium might convert acetate into valine. Assume an abundant supply of all needed cofactors and nitrogen donors.
April 14, 2001, 1:30 AM - 4:30 PM Working Page Working Space - (Not to be Marked)