The aim of this module is to develop the capacity of the student to undertake basic analytical and preparative procedures in the biochemistry laboratory, and to provide a foundation course in protein structure and enzymology
Safety practices in the biochemistry laboratory. Maintaining laboratory notebooks to GLP standard. Système International units. The periodic table. Definition of element, atom and molecule. Ionic and covalent bonds. Preparation of solutions. Avogadro’s Number: the mole concept. Modes of expressing units of concentration of solute in solution. Molarity and molality. Percent concentration (w/v; v/v; w/w; mg percent), parts-per-million and parts-per billion. Inter-converting between units. Molarity of ions. Percent composition. Calculation of dilution factors. Accuracy, precision (within/between batch), sensitivity and specificity. Random and systematic error. Use of the calculator for basic statistical calculations.
Spectrophotometry. The nature of light. Definition of a chromophore. Beer-Lambert law. Structural studies of biomolecules: absorbance spectra (l-max) and isosbestic point. Components of the spectrophotometer: construction and operation. Practical use of extinction coefficients. The use of spectrophotometry to conduct protein assay measurements via (a) Absorbance 280 nm; (b) Absorbance 205-230 nm; (c) Chemical protein assay (Biuret, Folin-Lowry, Bicinchoninic Acid, Bradford).
Structure of the water molecule: physical properties and influence on life processes. Water as a solvent. Dissociation of the water molecule. The Ion Product of water. Definitions of acids and bases. The concept of pH and pKa; calculation of hydrogen ion concentration and pH. The Henderson-Hasselbalch equation. Buffers: choice and preparation. Physiological relevance of pH. Buffer systems in living organisms (proteins, phosphate, bicarbonate). Medical relevance of pH balance.
Outline amino acid structure and classification.
Outline and discuss the levels of organisation used to describe the structure of proteins and recognise the relationship between structure and function and the functional diversity of proteins.
Explain the factors that determine the rate of an enzyme catalysed reaction and describe how the rate of an enzyme catalysed reaction is measured.
Apply their knowledge of enzyme assay to determining the activity of alkaline phosphatase and measuring the Km and Vmax of this enzyme.
Describe the properties of allosteric enzymes and explain the role played by these in regulating metabolism.
Recognise the influence of pH and temperature on protein stability
Laboratory Programme : Practicals will be selected from the following list:
Determination of the absorbance spectra of biomolecules (blue dextran, haemoglobin and NADH)
Determination of the molar absorptivity of para-nitrophenol and study of the precision and accuracy associated with this measurement.
Use of the pH meter in the preparation of buffer solutions, and determination of the isosbestic point for methyl red.
Determination of protein concentration using the Bradford method.
Miniaturization of biochemical assays: determination of protein concentration using a microplate version of the BCA assay.
Effect of pH and temperature on Alkaline phosphatase activity
Determination of Km and Vmax for Alkaline phosphatase
Isomers in Biochemistry
Basic Laboratory Procedures & Calculations
Safety practices in the biochemistry laboratory. Maintaining laboratory notebooks to GLP standard. Système International units. The periodic table. Definition of element, atom and molecule. Ionic and covalent bonds. Preparation of solutions. Avogadro’s Number: the mole concept. Modes of expressing units of concentration of solute in solution. Molarity and molality. Percent concentration (w/v; v/v; w/w; mg percent), parts-per-million and parts-per billion. Inter-converting between units. Molarity of ions. Percent composition. Calculation of dilution factors. Accuracy, precision (within/between batch), sensitivity and specificity. Random and systematic error. Use of the calculator for basic statistical calculations.
Spectrophotometry and Protein Assay
Spectrophotometry. The nature of light. Definition of a chromophore. Beer-Lambert law. Structural studies of biomolecules: absorbance spectra (-max) and isosbestic point. Components of the spectrophotometer: construction and operation. Practical use of extinction coefficients. The use of spectrophotometry to conduct protein assay measurements via (a) Absorbance 280 nm; (b) Absorbance 205-230 nm; (c) Chemical protein assay (Biuret, Folin-Lowry, Bicinchoninic Acid, Bradford).
Water and pH
Structure of the water molecule: physical properties and influence on life processes. Water as a solvent. Dissociation of the water molecule. The Ion Product of water. Definitions of acids and bases. The concept of pH and pKa; calculation of hydrogen ion concentration and pH. The Henderson-Hasselbalch equation. Buffers: choice and preparation. Physiological relevance of pH. Buffer systems in living organisms (proteins, phosphate, bicarbonate). Medical relevance of pH balance.
Protein Structure
Outline amino acid structure and classification.Outline and discuss the levels of organisation used to describe the structure of proteins and recognise the relationship between structure and function and the functional diversity of proteins.
Enzymology
Explain the factors that determine the rate of an enzyme catalysed reaction and describe how the rate of an enzyme catalysed reaction is measured. Apply their knowledge of enzyme assay to determining the activity of alkaline phosphatase and measuring the Km and Vmax of this enzyme.Describe the properties of allosteric enzymes and explain the role played by these in regulating metabolism.Recognise the influence of pH and temperature on protein stability
Laboratory Practicals
Determination of the absorbance spectra of biomolecules (blue dextran, haemoglobin and NADH)Determination of the molar absorptivity of para-nitrophenol and study of the precision and accuracy associated with this measurement.Use of the pH meter in the preparation of buffer solutions, and determination of the isosbestic point for methyl red.Determination of protein concentration using the Bradford method.Miniaturization of biochemical assays: determination of protein concentration using a microplate version of the BCA assay.Effect of pH and temperature on Alkaline phosphatase activityDetermination of Km and Vmax for Alkaline phosphataseIsomers in Biochemistry
Learning outcomes will be achieved through a combination of lectures, practicals, problem solving exercises and self-directed learning
| Module Content & Assessment | |
|---|---|
| Assessment Breakdown | % |
| Formal Examination | 60 |
| Other Assessment(s) | 40 |