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Exercise 3:

Exercise 3:. Enzymes. Announcements. Post Lab 3 and Pre Lab 4 are due by the time your lab meets next. LNA Enzymes is assigned today, and due next week within the first 5 minutes of your lab period. Exercise 2B:.

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Exercise 3:

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  1. Exercise 3: Enzymes

  2. Announcements • Post Lab 3 and Pre Lab 4 are due by the time your lab meets next. • LNA Enzymes is assigned today, and due next week within the first 5 minutes of your lab period.

  3. Exercise 2B: • Experimental Plates: Examine your plates and observe the type of bacteria or fungal growth that appears on each. • Streak Plates: Examine the colonies; TA will be around to assign your “Skills” score • Complete your LNA and turn it in as directed by your TA.

  4. Exercise 3A: Spectrophotometry • Goals: • Understand the process by which spectrophotometry can be used to quantify experimental results. • Develop skills taking measurements using a spectrophotometer.

  5. Spectrophotometry Measurement of light absorption or transmission through a solution

  6. Types of photometers: Colorimeters and spectrophotometers measure the amount of light absorbed by solutions. Turbidimeters and nephelometers measure the light scattered by suspensions. Fluorimeters measure the fluorescence produced by absorbed light.

  7. Light through a solution: • Example ONP (o-nitrophenol) • Absorbs blue light and allows yellow light to pass through. • Solution therefore appears yellow.

  8. Absorption Spectrum Absorption spectrum of ONP A plot of the relative amount of light absorbed by a compound as a function of the wavelength

  9. Components of a Typical Spectrophotometer

  10. Beer’s Law Mathematical expression relating concentration, length of the light path through the solution, and the light absorbed by a solution. Refer to Lab Manual pgs. 50-51

  11. 3A Techniques: Using colored water, take measurements using a spectrophotometer. Keep cuvettes free from fingerprints. Align cuvette correctly each time you take a measurement.

  12. 3B: Enzymes • Goals: • Describe the principles of enzymatic reactions • Use the principes of spectrophotometry to determine the concentration of the product of an enzyme-catalyzed reaction. • Determine the effect of ß-galactosidase concentration on the rate of cleavage of ONPG.

  13. Introduction: Enzymes increase the rate of reactions, but do not allow reactions to occur that could not occur otherwise.

  14. There are two ways to increase the rate of a chemical reaction • Increase the average kinetic energy by raising the temperature, or • Lower the activation energy by adding a catalyst

  15. Catalyzed Reaction Reach a Maximum Rate

  16. Enzyme Regulation

  17. -galactosidase O-nitrophenyl--D-galactopyranoside (ONPG)

  18. Hypothesis Generation • A yellow solution is produced as o- nitrophenolate is produced Identify one characteristic you expect to change as you add ONPG, buffer, and enzyme

  19. If ONPG is catalyzed by -galactosidase, and I add the enzyme in various amounts, the products of o-nitrophenolate (yellow color) will differ as well • Rephrase your speculation to the if, then format

  20. Independent Variable Amount of -galactosidase

  21. Dependent Variable OD420 reading of o-nitrophenolate produced

  22. Controls ONPG + Buffer, but no enzyme added and Buffer + enzyme, but no ONPG added

  23. The Experiment: Take measurements of various amounts of ONPG catalyzed by -galactosidase

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