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Enzymes and Enzymatic Reactions

Enzymes and Enzymatic Reactions. A Sample Experiment. Observation: Lit Search. A Literature Search allows you to discover information about what already has been discovered about your system of interest. Observation: Lit Search.

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Enzymes and Enzymatic Reactions

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  1. Enzymes and Enzymatic Reactions A Sample Experiment

  2. Observation: Lit Search A Literature Search allows you to discover information about what already has been discovered about your system of interest.

  3. Observation: Lit Search From your search, you can make a (third person) OBSERVATIONabout your system.

  4. Observation: Lit Search This week we will consider the effect of NaClon catalase. Let’s pretend we’ve already done a literature search.

  5. Observation: Lit Search From our literature search, we learned that… • Increased NaCl concentration increasesperoxidase activity in the halophytic archaen, Halobacterium halobium. (Brown-Peterson 1993)

  6. Observation: Lit Search From our literature search, we learned that… • In rice (Oryza sativa), salt stress was induced by immersing the plant’s roots in 0.1M NaCl for 48 hours. (Swapna 2003)

  7. Observation: Lit Search From our literature search, we learned that… • Many plants show increased peroxidase activity when experiencing salt stress.

  8. Observation: Lit Search From our literature search, we learned that… • In yeast, stress response was induced by 45 minutes of exposure to 0.3M NaCl. (Lewis 1995)

  9. Question Based on these observations, we might ask… “How would a change in NaCl concentration (for example, from 0.0M to 0.3M) affect yeast’s rate of hydrogen peroxide catalysis?"

  10. Overall Hypothesis Phrased as an overall hypothesis, one might posit… “Salt stress will affect the rate of hydrogen peroxide catabolysis in yeast (Saccharomycescereviseae).”

  11. Experimental Hypotheses Phrased as a two-tailed statistical hypotheses, this becomes HO – The rate of oxygen production will not differ between control yeast and yeast subjected to salt stress. HA – The rate of oxygen production will differ between control yeast and yeast subjected to salt stress.

  12. Experimental Hypotheses The null hypothesis could be stated: HO – The rate of oxygen production will not differ between control yeast and yeast subjected to salt stress.

  13. Experimental Hypotheses The two-tailed alternative hypothesis : HA– The rate of oxygen production will differ between control yeast and yeast subjected to salt stress.

  14. Experimental Hypotheses The one-tailed alternative hypothesis : HA– The rate of oxygen production in salt stressed yeast will be higher (or lower) than that of control yeast.

  15. Prediction We know (from our literature search) that increased NaCl concentration increases catalase activity in multiple, distantly related species.

  16. Prediction Therefore, a logical prediction would be: Increasing NaCl concentration in a yeast suspension from 0M to 0.3M will increase the rate of the peroxidase reaction.

  17. Experimental Design Based NaClsolution concentrations from the literature search, we will compare the rate of O2 production (a measure of H2O2 catabolysis) in • untreated yeast • yeast incubated for 30 minutes in 0.3M NaCl solution (to induce salt stress).

  18. Experimental Design • The type of data collected will be reaction rate: O2 produced/minute. • Control and treatment groups will each consist of multiple experimental runs. • Mean reaction rates of control and treatment groups will be compared.

  19. Statistical Analysis Recall the types of data.

  20. Statistical Analysis Will your data be • qualitativeorquantitative? • discrete/ordinal or continuous? • parametric or non-parametric?

  21. Planning Your Experiment Step One: develop a solid plan. • All calculations needed for mixing reagents should be done in advance . • The protocol plan must be complete before the experiment is begun.

  22. Planning Your Experiment Step Two: Know Your Reagents. 1. Stock yeast suspension: • 70g yeast/L of pH 7 sodium phosphate buffer solution.

  23. Planning Your Experiment Step Two: Know Your Reagents. 2. Stock H2O2 solution • 33mL of 30-volume (9.1%) hydrogen peroxide in 1L of pH 7 sodium phosphate

  24. Planning Your Experiment Step Three: Calculate Your Quantities. You will run six trials • three treatment • three control

  25. Planning Your Experiment Step Three: Calculate Your Quantities. Yeast suspension: • each trial requires 10mL of yeast suspension • Six trials 60mL yeast suspension. • Take no more than 70mL to allow for measurement error. Don’t waste!

  26. Planning Your Experiment Step Three: Calculate Your Quantities. Hydrogen Peroxide (H2O2) solution: • Each trial requires 20mL • Six trials  120mL H2O2solution • Take no more than 135mL to allow for measurement error. Don’t waste!

  27. Planning Your Experiment Step Three: Calculate Your Quantities. Sodium chloride (NaCl) 1M solution: • Yeast will incubate for 30 minutes to allow the yeast to react to the increased salt concentration.

  28. Planning Your Experiment Never measure reagents in • beakers • flasks THEY ARE NOT ACCURATE.

  29. Planning Your Experiment Only graduated cylinders or syringes are accurate enough for proper measurement of reagents in any experiment.

  30. Planning Your Experiment Know how to calculate MOLARITY: moles of solute /liters of solution. (One mole = 6.02 x 10 23particles)

  31. Planning Your Experiment A one molar (1M) solution contains 6.02 x 10 23 molecules of solute/L of solution.

  32. Planning Your Experiment If you have a stock solution of known molarity and you want your final yeast suspension to be a different molarity, use the following formula.

  33. Planning Your Experiment [stockNaCl (moles/L)] xstockNaCl volume (L) = [desiredNaCl in yeast suspension (moles/L)] x desired final volume (L)

  34. Planning Your Experiment For example, if you want • 0.3M NaCl in • 15mL of yeast suspension • and have a 1M solution of NaCl solve for x: (1 mole/L) (x) = (0.3mole/L) x (15mL)

  35. Planning Your Experiment x = 0.3moles/L x 15mL 1 mole/L

  36. Planning Your Experiment x= ?

  37. Planning Your Experiment x= 4.5mL of stock NaCl solution

  38. Controls are Critical! REMEMBER: • Your treatment and control vessels must contain exactly the samevolumesof solution.

  39. Controls are Critical! REMEMBER: • Your treatment and control vessels must be subjected to exactly the same conditions

  40. Controls are Critical! REMEMBER: • Your treatment and control vessels must contain exactly the same componentsEXCEPTfor the one variable you are testing

  41. Controls are Critical! If you add a particular volume of NaCl to your treatmentvessel, you must add exactly the same amount of DI water to the controlvessel.

  42. Controls are Critical! If you incubate your treatmentsample for a particular amount of time, you must incubate your controlsample for exactly the same amount of time.

  43. When you are finished • Pour all left-over solutions into the waste solution container EXCEPT leftover DI water. • Leftover, uncontaminated DI water can be poured down the sink.

  44. When you are finished • All used syringes should be discarded in the trash. • Do not place used syringes back on your tray.

  45. Time to Begin Open your online lab manual and follow the step-by-step instructions. Ready. Set. Go!

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