Enzymes and Enzymatic Reactions A Sample Experiment Observation

Enzymes and Enzymatic Reactions A Sample Experiment

Observation: Lit Search Your Literature Search allowed you to discover some of what is known about our system of interest, the peroxidase known as catalase.

Observation: Lit Search You can consider findings from your literature search to be an OBSERVATION about your system.

Observation: Lit Search A literature search reveals that the effect of salt stress on peroxidases has received quite a bit of interest from investigators.

Observation: Lit Search The physiological effect of salt stress on plants and other organisms is of great ecological and economic importance.

Literature Search Reveals. . . 1. Increased Na. Cl concentration increases peroxidase activity in the halophytic archaen, Halobacterium halobium. (Brown-Peterson 1993)

Literature Search Reveals. . . 2. In rice (Oryza sativa), salt stress was induced by immersing the plants' roots in 0. 1 M Na. Cl for 48 hours. (Swapna 2003)

Literature Search Reveals. . . 3. Many plants show increased peroxidase activity when experiencing salt stress. This is because environmental stress can increase production of potentially damaging Reactive Oxygen Species (ROS) such as hydrogen peroxide (H 2 O 2).

Literature Search Reveals. . . 4. In yeast, stress response was induced by 45 minutes of exposure to 0. 3 M Na. Cl. (Lewis 1995)

Question Based on these observations, we might ask… “How would a change in Na. Cl concentration (for example, from 0. 0 M to 0. 3 M) affect yeast’s rate of hydrogen peroxide catalysis? "

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

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.

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

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.

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

Prediction Therefore, a logical prediction would be: Increasing Na. Cl concentration in a yeast suspension from 0 M to 0. 3 M will increase the rate of the peroxidase reaction.

Experimental Design Based on Na. Cl solution concentrations from the literature search, we will compare the rate of O 2 production (a measure of H 2 O 2 catabolysis) in – untreated yeast – yeast incubated for 30 minutes in 0. 3 M Na. Cl solution (to induce salt stress).

Experimental Design 1. The type of data collected will be reaction rate: O 2 produced/minute. 2. Control and treatment groups will each consist of multiple experimental runs. 3. Mean reaction rates of control and treatment groups will be compared.

Statistical Analysis Recall the types of data.

Statistical Analysis Will your data be • qualitative or quantitative? • discrete/ordinal or continuous? • parametric or non-parametric? Use the statistics flow chart.

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.

Planning Your Experiment Step Two: Know Your Reagents. 1. Stock yeast suspension: – 70 g yeast/L of p. H 7 sodium phosphate buffer solution.

Planning Your Experiment Step Two: Know Your Reagents. 2. Stock H 2 O 2 solution – 33 m. L of 30 -volume (9. 1%) hydrogen peroxide in 1 L of p. H 7 sodium phosphate

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

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

Planning Your Experiment Step Three: Calculate Your Quantities. Hydrogen Peroxide (H 2 O 2) solution: • Each trial requires 20 m. L • Six trials 120 m. L H 2 O 2 solution • Take no more than 135 m. L to allow for measurement error. Don’t waste!

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

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

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

Planning Your Experiment Know how to calculate MOLARITY: moles of solute /liters of solution. (One mole = 6. 02 x 10 23 particles)

Planning Your Experiment A one molar (1 M) solution contains 6. 02 x 10 23 molecules of solute/L of solution.

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.

Planning Your Experiment [stock Na. Cl (moles/L)] x stock Na. Cl volume (L) = [desired Na. Cl in yeast suspension (moles/L)] x desired final volume (L)

Planning Your Experiment For example, if you want – 0. 3 M Na. Cl in – 15 m. L of yeast suspension – and have a 1 M solution of Na. Cl solve for x: (1 mole/L) (x) = (0. 3 mole/L) x (15 m. L)

Planning Your Experiment x = 0. 3 moles/L x 15 m. L 1 mole/L

Planning Your Experiment x = ?

Planning Your Experiment x = 4. 5 m. L of stock Na. Cl solution

Controls are Critical! REMEMBER: • Your treatment and control vessels must contain exactly the same volumes of solution.

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

Controls are Critical! REMEMBER: • Your treatment and control vessels must contain exactly the same components EXCEPT for the one variable you are testing

Controls are Critical! If you add a particular volume of Na. Cl to your treatment vessel, you must add exactly the same amount of DI water to the control vessel.

Controls are Critical! If you incubate your treatment sample for a particular amount of time, you must incubate your control sample for exactly the same amount of time.

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.