Human Physiology Lab (Biol 236L) Digestive Physiology: Amylase hydrolysis of starch

Transcription

1 Human Physiology Lab (Biol 236L) Digestive Physiology: Amylase hydrolysis of starch Introduction Enzymes are proteins composed of amino acid building blocks. Enzymes catalyze or increase the rate of metabolic reactions, allowing them to occur at temperatures within the range tolerated by living cells. Thus, enzymes are an important group of functional molecules that regulate metabolic reactions in all living cells from bacteria to plants to animals. Enzymes, like all proteins, have a specific three-dimensional shape that results from the coiling and twisting of the amino acid chains. Each type of enzyme has its own specific shape, and this shape is critical to the enzyme s function. An active site exists somewhere on the surface of the enzyme, and the substrate (or reactant) molecules of the reaction bind to the active site. The resulting enzyme-substrate complex facilitates the bond formation (or breaking; enzymes are involved in breaking down molecules as well as building them), resulting in the final product. See Figure 2.1 for an example of sucrose breakdown. One well-known enzyme, amylase, is found in both plants and animals. (Note: most enzyme names end in the suffix -ase). Amylase begins the process of starch breakdown. Recall that starch is a common plant polysaccharide, composed of repeating glucose monomers. Starch is common in our diets, and in order to digest starch for energy, we must first break the polymer down into smaller subunits for transport to the cells. Starch breakdown starts in your mouth with salivary amylase. The amylase begins cleaving the starch molecule into maltose subunits (maltose is a disaccharide, consisting of two glucose monomers). The breakdown of starch is a good example of a hydrolysis reaction the insertion of a water molecule to lyse (break apart) a larger molecule into smaller subunits. Figure 2.1. Descriptive example of an enzyme (sucrose) breaking up the substrate (sucrose) into end products of glucose and fructose. Note that the enzyme is unchanged after the reaction. 1

2 To achieve our objectives we will test the effect of temperature, ph, substrate concentration, and enzyme concentration on the efficiency of salivary amylase. We will use hydrolysis time as an indicator of enzyme efficiency. Equation showing the hydrolysis of starch: Experiments: The class will be divided into 4 6 teams. Each team will perform only one experiment. At the end of lab you will present your data and discuss what each team has done. You will be responsible not only for your results but for the results of all other teams. In each experiment you will be testing for the efficiency of salivary amylase, but under different experimental conditions. Please note that in all of the above experiments, hydrolysis time is the dependent variable. Starch in the presence of iodine (indicator solution) turns dark blue or black. As long as your solution contains starch it will remain bluish-black in color. When the salivary amylase has completely broken down the starch (polysaccharide) into maltose (disaccharide) you will see a light yellow color when iodine is added indicating that the hydrolysis reaction is complete. For sanitary reasons one person from each group will be designated as the collector of saliva (spitter) and pipetter. The collector should produce about 5 ml of saliva (one inch in the plastic falcon tube). Dilute this saliva with 5 ml distilled water. This diluted amylase (spit) solution is your enzyme solution. Experiment 1. The effect of temperature on hydrolysis time. Measure the effect of temperature on the efficiency of salivary amylase. One person out of the group should spit and pipette. Each of the remaining members will select a temperature to test and complete the following: Procedure: 1. Add 50 ml starch to the flask. 2. Adjust the temperature inside the flask by placing it in hot H 2 O, cold H 2 O, or ice. Be sure the temperature (15 º, 30 º, 45 º, or 70 º C) remains constant throughout the entire experiment. 3. When the temperature is stable add 1.0 ml enzyme solution (diluted saliva) to your flask, and start the timer. Do not stop the timer until the experiment is complete! 4. Every 2 minutes add a dropperful of solution in the spot plate and add 1 drop of Iodine. 5. Record the time it takes for the reaction to be completed (hydrolysis time) in Table 1. Put Experiment 2. The effect of ph on hydrolysis time. Here you will test the effect of ph on the efficiency of salivary amylase. One person out of the group should spit and pipette. Each of the remaining members should choose a ph (4.0, 6.4, 8.0, or 9.0) to work with and do the following: 2

3 Procedure: 1. Add 25 ml of your designated ph buffer solution to a flask. 2. Add 0.5 ml enzyme (saliva). 3. At this point add 25 ml starch to the flask and start your time clock. Do not stop the timer until the experiment is complete! 4. Every 2 minutes put a dropperful of solution in the spot plate and add 1 drop of Iodine. 5. Record the time it takes for the reaction to be completed (hydrolysis time) in Table 1. Put Experiment 3. The effect of substrate concentration on hydrolysis time. Measure the effect of substrate (starch) concentration on the efficiency of salivary amylase. One person out of the group should spit and pipette. The remaining members should proceed to set up the test as follows: Procedure: 1. Add 25 ml of H 2 O to each of 4 flasks. 2. Add 25 ml of starch to the first flask ONLY (a 1:2 dilution). 3. Stir the flask. 4. Take 25 ml of solution from the first flask, add it to the second flask and mix (a 1:4 dilution). 5. Take 25 ml from second and add to the third flask, mix (a 1:8 dilution). Take 25 ml from the third and add to the fourth flask and mix (a 1:16 dilution). Discard 25 ml from the 4th flask into the sink. 6. Make sure you have diluted the saliva with 5 ml H 2 O, as previously instructed. 7. Add 0.1 ml saliva to each flask and start the time clock. Do not stop the timer until the experiment is complete! (You may find it easier to begin with only 2 flasks at a time. This means add saliva only to flask #1 and #2 and start the timer, then begin work on flasks #3 and #4). 8. Every 2 minutes put a dropperful of solution in the spot plate and add 1 drop of Iodine. 9. Record the time it takes for the reaction to be completed (hydrolysis time) in Table 1. Put Experiment 4. The effect of enzyme concentration on hydrolysis time. Measure the effect of enzyme concentrations on the efficiency of salivary amylase. One person out of the group should spit and pipette. The remaining members should proceed to set up the test as follows: Procedure: 1. Add 20 ml H 2 O to each flask. 2. Add 5.0 ml enzyme (saliva) to the first flask ONLY! (a 1:5 dilution). (Be sure the saliva is diluted with 5.0 ml H 2 O, as previously instructed). 3. Take 5.0 ml solution from flask 1 and add it to flask 2 and mix (a 1:25 dilution). Take 5.0 ml from the flask 2 and add to the flask 3, mix (a 1:125 dilution). Take 5.0 ml from the flask 3 and add to the flask 4, mix (a 1:625 dilution). Take 5.0 ml from the flask 4 and discard it into the sink. 4. Add 25 ml of starch to all of the flasks and start your time clock. Do not stop the timer until the experiment is complete! (You may find it easier to begin with only 2 flasks at a time. This means add the 25 ml of starch to flasks 1 and 2 only and begin timing these two only. After you have these running smoothly then begin with flasks 3 and Every 2 minutes put a dropperful of solution in the spot plate and add 1 drop of Iodine. 6. Record the time it takes for the reaction to be completed (hydrolysis time) in Table 1. Put 3

4 Record and Graph the Class Data Record the data, where appropriate, in Table 1 below. Then add your data to the Excel spreadsheet provided by your instructor. You will graph the class summary results at the end of this lab. Identify the dependent and independent variables, and the scales for the X- and Y-axes before you begin. Table 1. Hydrolysis times of salivary amylase under different conditions. Temperature C ph Substrate dilution 1:16 1:8 1:4 1:2 Enzyme dilution 1:625 1:125 1:25 1:5 4

5 Time (minutes) Time (minutes) Temperature C ph 5

6 Time (minutes) Time (minutes) 1:16 1:8 1:4 1:2 Substrate Dilution 1:625 1:125 1:25 1:5 Enzyme Dilution 6

7 Questions: The general scheme for an enzyme reaction is: E + S > ES > E + P 1. Name the enzyme. 2. Name the substrate of the reaction. 3. Name the product. 4. What happens to the product after the reaction takes place in your body? 5. This hydrolysis reaction is an example of the use of an enzyme for which process? synthesis photosynthesis digestion secretion 6. What is meant by optimal conditions for a reaction? 7. Which TWO experiments exhibited an optimum and HOW DO YOU KNOW? 8. What is the effect of increased substrate concentration on the hydrolysis time with a constant amount of enzyme? 9. What is the effect of increased enzyme concentration on the hydrolysis time with a constant amount of substrate? 10. How could you tell that the reactions were completed? 7