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Unformatted text preview: Exercise 3. Enzymes 39 EXERCISE 3 ANALYSIS OF ENZYME PROPERTIES AND RESTRICTION ENDODUCLEASES Nothing in biology makes sense except in the light of evolution. Theodosius Dobzhansky, The American Biology Teacher 1973 * COLORIMETRIC ENZYME ASSAY FOR TYROSINASE * RESTRICTION DIGEST OF LAMBDA DNA Equipment and supplies needed Colorimetric enzyme assay spectrophotometer mushrooms blender Erlenmeyer, funnel and cheesecloth 100mM phosphate buffer pH 6.0 vials for enzyme pipettors 10 ml (1) and P20, and P200 test tubes and racks 20mM phosphate buffer pH 6.0 (on ice) substrate solution (2mM DOPA) parafilm heat blocks at 40, 60, 80. And 90C timers 0.5 M SDS Computers for graphing data Restriction Digest of DNA Lambda DNA (0.4 g/l) MspI 2.5 units/l HindIII 2.5 units/l microfuge tubes microfuge racks heat blocks 37C P20 pipetman and tips Microcentrifuge MSPI and HINDIII 2X buffer Plant Genetics F1 plants The purposes of this exercise to investigate the properties of two enzymes. First, tyrosinase, which oxidzes tyrosine and, second, two restriction endonucleases which selectively degrade DNA. The cells of all organisms carry out thousands of different chemical reactions every minute of the day and night for as long as the organism survives. Most of these reactions require catalysts , which in biological systems are called enzymes. Catalysts are defined as agents Exercise 3. Enzymes 40 that speed up chemical reactions by lowering the activation energy required. In practice this means that in the presence of a catalyst, reactions can occur at greater speeds and at much lower temperatures than would normally be necessary. Enzymes are thus a critical component of all cells and have enabled the enormously complex variety of life forms to thrive on our earth at temperatures we normally experience. The majority of the enzymes characterized thus far are proteins. Recently some RNA molecules, known as ribozymes, have also been found to have catalytic properties. This important discovery has revolutionized our view of how life evolved on Earth and we will learn more about this in EEMB 3, the last course in the introductory series that deals with diversity of life. In this exercise you will investigate the properties of protein catalysts. The simplest description of what enzymes do can be diagrammed as: SUBSTRATE PRODUCT ENZYME Although this diagram omits many of the most interesting properties of enzymes, it points out through the use of the term substrate that the reactants provide a surface on which the enzymes act. It also points out that enzymes are not used up in the reaction, nor do they alter the overall energetics or stoichiometry of it....
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