Module Framework: Biochemistry 314 - 2004

  1. Name and code of the module

    Biochemistry 314 - Advanced Biochemical Topics I
    11053 314

  2. Lecturers and contact details

    Module convenor: Protein biochemistry: Molecular cell physiology:
    Prof JM Rohwer Dr M Rautenbach Prof JL Snoep
    Room A108, JC Smuts Building Room A115, JC Smuts Building Room A114, JC Smuts Building
    Tel.: 021 808-5843 Tel.: 021 808-5872 Tel.: 021 808-5844
    Email: jr@sun.ac.za Email: mra@sun.ac.za Email: jls@sun.ac.za
     
    Practical:    
    Dr AC Swart Ms Z Allie Ms RP Louw
    Room A120, JC Smuts Building Room A117, JC Smuts Building Lab A139, JC Smuts Building
    Tel.: 021 808-5864 Tel.: 021 808-5883 Tel.: 021 808-5877
    Email: acswart@sun.ac.za Email: zyno@sun.ac.za Email: rpl@sun.ac.za

  3. Objectives of the module

    We strive to develop the following skills in the students:

    • factual knowledge of: protein purification and the determination of protein structure and function; practical techniques used in protein purification; functional description of metabolic regulation, appropriate thermodynamic and kinetic principles, and why the classical description is insufficient;
    • unlocking and processing subject information from electronic and traditional (printed) sources;
    • numerical skills in biochemical calculations;
    • visual conceptualisation skills (interpretation of graphs);
    • problem analysis, problem solving and lateral thinking (bringing together different concepts to solve a problem);
    • practical skills in mastering certain techniques of protein purification;
    • logical analysis and critical evaluation of experimental data from protein purification and analysis;
    • writing skills in terms of the logical presentation and formulation of an argument, to be applied in problem-based tasks and in reports on experimental data;
    • general laboratory skills, i.e. time planning, time management, group/team work, sense of responsibility, computer literacy.

  4. Outcomes of the module

    Section A: Advanced protein biochemistry

    After completing this section, students should, with the required knowledge and skills, be able to:

    1. name the different fractionation techniques used in tissue preparation, and explain their basic principles;
    2. explain the effect of external factors such as pH, temperature, ionic strength, organic solvents and detergents on protein conformation and solubility;
    3. explain how solubility/stability, size, net charge and binding specificity are used in protein isolations;
    4. explain the basic principles of the following separation techniques: ultracentrifugation, salting out and salting in, electrophoresis, SDS-PAGE, iso-electric focussing, gel permeation chromatography, ion exchange chromatography, normal phase chromatography, reverse phase chromatography;
    5. name the various methods that can be used to determine the mass of a protein, and explain how each of them works;
    6. discuss the different levels of protein structure with reference to examples from muscle proteins;
    7. briefly explain the thermodynamics of protein folding and denaturation by referring to the role of non-covalent interactions and the hydrophobic effect;
    8. name the different methods and chemical reactions used to determine protein structure, and discuss them with reference to specific examples;
    9. discuss the basic principles of the following spectrophotometric techniques: light spectrophotometry, circular dichroism, infrared spectrophotometry, nuclear magnetic resonance spectrometry, X-ray crystallography;
    10. discuss the specific techniques used to determine each of 2o, 3o and 4o structures;
    11. comprehensively discuss the most important proteins in muscle contraction with regard to their structure (2o, 3o and 4o and higher order)-function relationships and their role in the process of muscle contraction;
    12. given the critical parameters in ultracentrifugation, to calculate radians/second, revolutions per minute, the k-factor of the rotor, the centrifugation force and the relative centrifugal force;
    13. given the data from an experiment (SDS-PAGE, gel permeation chromatography or mass spectrometry) to determine molecular mass, analyse the data critically with regard to the Mr of the protein and its purity;
    14. given chromatography data (gel permeation, ion exchange or HPLC) of proteins/peptides, to determine the chromatographic parameters, as well as the character and purity of the protein/peptide;
    15. given data from a separation process of proteins or peptides, analyse it with regard to the character and purity of the protein or peptide;
    16. suggest and/or justify a protocol for the isolation of a protein of which the character is known (e.g. a known muscle protein);
    17. given the amino acid sequence of different peptides, predict and explain their order of elution/retention on a specific gel permeation, ion exchange, silica and C18*-silica column (*C2, C4 or C8);
    18. suggest and/or justify a protocol for the determination of the amino acid sequence of a peptide;
    19. given the data from a sequence determination experiment of a peptide, deduce its amino acid composition, Mr and sequence;
    20. given a protein's amino acid composition, deduce its solubility properties;
    21. given spectrophotometric data (UV/Vis, CD or IR spectra) of a protein/peptide, analyse it critically and make deductions about the structure and/or concentration of the protein/peptide;
    22. suggest and/or justify a protocol for the characterisation of a protein's 2o, 3o and 4o structure;
    23. critically evaluate and explain data on isolated muscle proteins;
    24. retrieve subject information on protein purification from both electronic and traditional sources, process it and formulate a logical argument and layout in writing in a task.

    Section B: Molecular cell physiology

    After completing this section, students should, with the required knowledge and skills, be able to:

    1. understand and explain the classical (textbook) view of metabolic regulation and its shortcomings;
    2. use (draw and interpret) rate characteristics as graphical tools for describing metabolic behaviour;
    3. understand, describe and apply in calculations the concept of DG as the driving force for a chemical reaction;
    4. distinguish between DG, DG0, DG', DG0', G and Keq;
    5. distinguish between the chemical and biochemical standard state;
    6. calculate equilibrium concentrations for substrates and products of a single reaction or of two coupled reactions, given Keq or DG0 values and initial conditions;
    7. distinguish between open and closed systems;
    8. distinguish between equilibrium and the steady state;
    9. apply in calculations the reversible Michaelis-Menten equation with or without uncompetitive product inhibition;
    10. use the Haldane relationship to transform the reversible Michaelis-Menten equation;
    11. define the meaning of each term in the reversible Hill equation (with and without allosteric modifiers) and apply this equation in calculations;
    12. distinguish between parameters and variables in a metabolic system;
    13. define control coefficients, elasticity coefficients and response coefficients mathematically, graphically and in words;
    14. calculate elasticity and control coefficients from rate characteristics, and give a physical interpretation of the results;
    15. explain the factory analogy of supply and demand for metabolic systems;
    16. show graphically what is the thermodynamic and kinetic contribution to the supply rate characteristic of a pathway for the following mechanisms: mass action, reversible Michaelis-Menten without and with uncompetitive product inhibition, reversible Hill;
    17. define functional differentiation in metabolic systems and deduce from a rate characteristic whether a system is functionally differentiated or not;
    18. define homeostasis in intermediate concentrations and deduce from a rate characteristic or from control coefficients whether an intermediate is homeostatically buffered;
    19. understand and explain the functional view of metabolic regulation, and how it addresses the shortcomings of the classical view.

    Practical

    After completing the practical module, the student should, with the required knowledge and skills, be able to:

    1. explain the following techniques in terms of their basic principles: gel filtration chromatography, cation exchange chromatography, spectrophotometric protein determinations, enzyme assays and SDS polyacrylamide electrophoresis (SDS-PAGE);
    2. perform the following techniques in the laboratory: gel filtration chromatography, cation exchange chromatography, spectrophotometric protein determinations, enzyme assays and SDS polyacrylamide electrophoresis (SDS-PAGE);
    3. apply the basic and practical principles of gel filtration and ion exchange chromatography to separate mixtures of proteins;
    4. calculate dilutions to be able to analyse protein concentration and enzyme activity data;
    5. apply the basic and practical principles of SDS-PAGE to determine the molecular mass of unknown proteins;
    6. draw up an experimental work protocol;
    7. process, analyse critically, and discuss experimental data from the practical;
    8. write an experimental report in the format and writing style of articles in biochemical journals;
    9. master general skills in laboratory work, i.e. time planning, time management, group/team work, sense of responsibility, and computer skills.

  5. Language specification

    A

  6. Compulsory study material

    • Voet, D. & Voet, J.G. (1995) Biochemistry, 2nd Edition, John Wiley & Sons Inc.
           OR
      Voet, D. & Voet, J.G. (2004) Biochemistry. Volume 1, Biomolecules, Mechanism Of Enzyme Action And Metabolism, 3rd Edition. John Wiley & Sons, Inc.
    • Wilson, K. & Walker, J. (2000) Principles and Techniques of Practical Biochemistry, Cambridge University Press, 5th edition.
    • Supplementary notes on Molecular Cell Physiology (will be handed out in class).
    • Optional text book: Lodish, H. et al. (2000) Molecular and Cell Biology, W.H. Freeman & Co., 4th edition.

  7. Learning opportunities

    Lectures: all lectures are in lecture hall A203, JC Smuts Building
      Monday 10:00-10:50 (3rd period)
      Tuesday 08:00-08:50 (1st period)
      Friday 11:00-11:50 (4th period)
     
    Practicals: Wednesday afternoon 14:00-17:00
      Timetable and groups will be announced by Dr AC Swart.
     
    Tutorials: Timetable will be announced later.

  8. Assessment

    The Biochemistry 314 module is assessed on the basis of continuous evaluation. There is no examination, and every assessment opportunity counts a percentage towards the final mark.

    1. Means of assessment
      • Written test in the mid-semester test series (on protein biochemistry)
      • Written test during exam time (on molecular cell physiology and protein biochemistry)
      • Assignment on protein biochemistry, to be completed by students in their own time
      • Assessment of practical (comprising: two written tests, rough reports and a final lab report)

    2. Place and time of assessment opportunities

      Theory

      • Mid-semester test on 24/03/2003 at 19h00 in the First-Year Chemistry Building (lower and upper lecture halls)
      • Test during exam time on 12/06/2003 at 09h00 (Saturday morning!) in the First-Year Chemistry Building

      Practial

      • Written test during Session 4, lecture hall A203, JC Smuts Building
      • Written test on 26/05/2003 at 14h00 in the First-Year Chemistry Building

    3. Turnaround time and format of feedback

      Where at all possible, assignments and tests will be marked within 3 weeks and handed back to the students.

    4. Calculation of class- and final mark

      The theory counts 70% and the practical counts 30% towards the final mark. There is a pass subminimum for both the theory and the practical components of the module. This means that if you obtain less than 50% for either the theory or the practical, you will fail Biochemistry 314 in its entirety!

      The theory mark (in total 70% of the final mark) is comprised as follows:

      • Mid-semester test (16%)
      • Test during exam time - protein biochemistry (16%)
      • Test during exam time - molecular cell physiology (23%)
      • Assignment on protein biochemistry (15%)

      The practical component counts 200 marks, which together account for 30% of the final mark:

      • Written test during Session 4 (20 marks)
      • Written test on 26/05/2003 (75 marks)
      • Explanation of experimental procedures/flow diagram, analysis and processing of results, rough reports (55 marks)
      • Final lab report (50 marks)

  9. Other special requirements

    If you have missed an assessment opportunity as a result of illness, you have to hand in a doctor's certificate to the module convenor within 7 days, in order to be admitted to a supplementary test.