Mechanism of Enzyme Action of Chymotrypsin
Chymotrypsin is a proteolytic enzyme which is synthesised in the pancreas and released into the digestive tract as an inactive proenzyme or zymogen. Once in the gut, it is activated by the removal of part of the peptide chain. Its biological role is to hydrolyse dietary protein in the small intestine. It cleaves the peptide bond on the C (carbonyl) side of the aromatic residues, phenylalanine, tyrosine and tryptophan, and some large hydrophobic residues e.g. methionine and leucine.
The students will explore the two-step reaction mechanism, the enzyme dependence of this reaction, the activation of the zymogen, chymotrypsinogen by trypsin. In this laboratory session the students are going to investigate the mechanism of action of chymotrypsin using the artificial substrate p-nitrophenylacetate (p-NPA).
DNA electrophoresis is a fundamental technique used in Molecular Biology to separate DNA fragments by size. In this experiment first year students digest lambda DNA with an unknown restriction enzyme (one of a possible 4) and separate the products by agarose gel electrophoresis. Based on the number and size of the fragments obtained they can identify their unknown enzyme. The sites on lambda for all 4 possible unknown restriction enzymes are provided so the expected fragment sizes for each possible digest can be predicted.
Enzymes I: Determination of acid phosphatase activity
This experiment looks at the area of enzyme kinetics using an acid phosphatase from potato. Students measure the breakdown of p-nitrophenyl phosphate by phosphatase, by measuring one of the products, p-nitrophenol (PNP). PNP can be measured spectrophotometrically as it is coloured (in alkaline solution). The other product is inorganic phosphate. Students finally graph the amount of p-nitrophenol produced in the reaction against time and use this graph to calculate the initial rate of the reaction, and then calculate the specific activity of the enzyme.
A chemical in the mouth
The basis behind these two laboratories is to describe the re-structuring of some old favourite Biology practical exercises: osmosis (cells and understanding water movement) and the action of the enzyme amylase (A chemical in the mouth). Basically these two exercises exist in numerous laboratory textbooks as standards and have become tacit ritualised practice (Ross et al., 2010). The restructuring described in this paper was done to create process of inquiry within a two- three-hour time period in a University practical laboratory. This process and the rationale of the redesign has many applications to other practical laboratories, which have for far too long been accepted as a ‘good’ way to do Biology.
Cells and understanding water movement
The cell is the basic unit of life and some basic processes, such as membrane function and protein synthesis, occur in the cells of all living organisms. This unit examines these processes and associated biological chemicals. The unit also examines phenomena such as cell replication, sex cell formation, inheritance, and cell metabolism in eukaryotes (animals, protists, fungi and plants). The biochemical capture of the sun’s energy (photosynthesis) is also investigated. The evolutionary links between cellular processes and the origin of life forms the framework for the unit.
Measurement of the Length of a Fatty Acid Molecule
It is often difficult to find laboratory activities which provide students with hands-on experience and data relating directly phenomena at the atomic or molecular level. This experiment is extremely easy to carry out, even for multiple labs for large cohorts of students, but still provides some direct data relating to the behaviour of single molecules of oleic acid. Analysis of the data requires students to; theorize about how individual molecules are behaving when added to water, apply an understanding of the polar nature of water molecules and how this affects the way it interacts with non-polar molecules, apply an understanding of how amphipathic molecules, in particular, behave in water, apply this understanding to how phospholipids behave in water leading to the formation of a bilayer, Develop and use skills in computations involving dilutions, area, volume, units of measurement, and exponents.
Cytogenetics: mitosis in the meristem of jonquil root tips
Students are able to observe the stages of mitosis under a compound microscope by preparing squashes of onion or jonquil root tips and staining with aceto-carmine. Students are to produce labelled diagrams of examples of each phase of mitosis from their preparation. Students also estimate the relative length of time of each phase of the cell cycle by recording the frequency with which they find each phase in regions where cell division is actively taking place. The frequency of a phase is an indication of the relative length of that phase. The cell cycles for onion and jonquil are about 16 hours.
The Structure and Function of Leaves
To introduce you to photosynthesis – the fundamental energy transforming process that occurs in green plants, and to the structure of the leaves that facilitate it. This process is essential for all life on earth as we know it. The experiment appears to meet all the criteria that students state they wish to be in a practical laboratory session (eg. Hands on, direct observation of a biological process, use of technical apparatus, quantitative measurement of a process taught in theory) but it consistently rates only average in student feedback.