Apr_07_237 - Marks 3 1. State which chemicals or products...

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Unformatted text preview: Marks 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. Name: ________________________________ Student Number: __________________ _______________________________________________________________________________________ THE UNIVERSITY OF MANITOBA April 16, 2007, 9:00 AM -12:00 PM Page 1 (of 4) Paper # 202 / 203 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. _______________________________________________________________________________________ NAD+ Ans_____________________________________________________________________ B) To form the polar phase for adsorption chromatography. Petroleum ether Ans_____________________________________________________________________ C) To act as the substrate in the assay used to measure lysozyme activity. Micrococcus lysodeikticus Ans_____________________________________________________________________ D) To act as an oxidizing agent in the assay used to measure blood glucose concentrations. Copper Sulfate Solution Ans_____________________________________________________________________ E) To form a coloured complex in the assay used to measure blood glucose concentrations. Arsenomolybdate Solution Ans_____________________________________________________________________ F) To precipitate protein and DNA in order to separate them from RNA. Potassium Hyrdoxide (KOH) Ans_____________________________________________________________________ 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. Experiment #10: Measurement of DNA and RNA from Calf Liver: - Assay name: Orcinol assay for RNA This uses bial's test for ribose: ribose Heat furfural Orcinol HCL Ferric ions Blue- Green Condensation Products Marks THE UNIVERSITY OF MANITOBA April 16, 2007, 9:00 AM -12:00 PM Page 2 (of 4) Paper # 202 / 203 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 PK PL PM PN PO PP MW 107,500 35,000 173,000 96,000 185,000 71,000 pI 10.2 4.8 7.3 6.2 8.6 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______________________________ PL PN Fraction 2A______________________________ PM, PO, PP 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. When proteins are passes through a CM-Sephadex column, positively charged proteins molecules will be held on the ion exchanger by exchanging with Na+ of the carboxyl group. Proteins that are negatively charged will be washed off the column since they are repelled by the -CH2COO- Marks 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______________________________ PO, PM PP Fraction 6B______________________________ THE UNIVERSITY OF MANITOBA April 16, 2007, 9:00 AM -12:00 PM Page 3 (of 4) Paper # 202 / 203 Biochemistry II Laboratory Section Final Examination MBIO / CHEM.2370 Examiner: Dr. A. Scoot _______________________________________________________________________________________ (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. Choice of gel type for gel filtration depends fo the shape and size of the molecules to be s eperated. For example with G-100, peptides with MWs below 4000 will all come off the column last and together since they will all go through all the pores. Proteins with MWs above 150,000 will come off the column first and together since they are all excluded from the pores. Molecules in between 4,000 and 150,000 go through the pores to a varying extent and are eluted in the order of decreasing MW. So the smaller ones elutes last. 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. Origin + - PM PO Marks 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? THE UNIVERSITY OF MANITOBA April 16, 2007, 9:00 AM -12:00 PM Page 4 (of 4) Paper # 202 / 203 Biochemistry II Laboratory Section Final Examination MBIO / CHEM.2370 Examiner: Dr. A. Scoot _______________________________________________________________________________________ 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. Marks 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. Name: ________________________________ Student Number: __________________ _______________________________________________________________________________________ THE UNIVERSITY OF MANITOBA April 16, 2007, 9:00 AM -12:00 PM Page 1 (of 4) Paper # 202 / 203 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. _______________________________________________________________________________________ 7 2. Describe using diagrams, the mechanism by which protein kinase and protein phosphatase control glycogen metabolism. Marks 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? THE UNIVERSITY OF MANITOBA April 16, 2007, 9:00 AM -12:00 PM Page 2 (of 4) Paper # 202 / 203 Biochemistry II Lecture Section Final Examination MBIO / CHEM 2370 Examiner: J. Stetefeld, P. Loewen _______________________________________________________________________________________ 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. Marks 18 5. Using compound structures and names and enzyme names or diagrams where necessary, describe: a) the role of sigma factor (￿) in transcription; _______________________________________________________________________________________ THE UNIVERSITY OF MANITOBA April 16, 2007, 9:00 AM -12:00 PM Page 3 (of 4) Paper # 202 / 203 Biochemistry II Lecture Section Final Examination MBIO / CHEM 2370 Examiner: J. Stetefeld, P. Loewen _______________________________________________________________________________________ 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. Marks 10 6. Using structural formulae and names of all intermediates and the names of enzymes and coenzymes, outline the steps required to convert CO2, 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. THE UNIVERSITY OF MANITOBA April 16, 2007, 9:00 AM -12:00 PM Page 4 (of 4) Paper # 202 / 203 Biochemistry II Lecture Section Final Examination MBIO / CHEM 2370 Examiner: J. Stetefeld, P. Loewen _______________________________________________________________________________________ 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? ...
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