Lab 4: Enzymes ANSWER KEY. 1. How could you test to see if an enzyme was completely saturated during an experiment? (2 pts)

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1 Lab 4: Enzymes ANSWER KEY Pre-Lab Questions 1. How could you test to see if an enzyme was completely saturated during an experiment? (2 pts) To test this, you d simply see if the reaction was still taking place. If the enzyme was completely depleted, the reaction could no longer occur. 2. List three conditions that would alter the activity of an enzyme. Be specific with your explanation. (2 pts) Temperature, salinity, and acidity are three conditions that affect enzyme activity. Specific examples include: Adding a salt packet to an enzyme would alter its activity--salt ions are often necessary for optimal enzyme activity, so too much or too little salt will slow down the rate. Enzyme activity increases with increasing temperature, until it becomes hot enough to denature the protein (enzymes are proteins), then the activity would cease. Enzymes have an optimum ph where they function the best. Higher or lower ph than the optimum slows the rate of activity. 3. Take a look around your house and identify household products that work by means of an enzyme. Name the products, and indicate how you know they work with an enzyme. (2 pts) Some examples include: contact lens cleaner (protease), dish soap (amylase), cheese (rennin), meat (papain), and starch products (amylase). Experiment 1: Enzymes in Food Post-Lab Questions

2 (Table - 2 pts) 1. What were your controls for this experiment? What did they demonstrate? Why was saliva included in this experiment? (2 pts) This experiment is looking for the presence of the enzyme amylase. A positive control would indicate the presence of amylase and so should be something that is KNOWN to contain amylase. A negative control would indicate no amylase, so should be a substance KNOWN to not contain amylase. As written, the positive control would be the ginger root because it is known to contain amylase. The presence of amylase is indirectly tested for by testing for the presence of starch with an IKI solution. Amylase breaks down starch into its monomers of the simple sugar glucose, so if amylase is present, there would be no starch because it would all be converted to sugars. If amylase is present on the paper towel it would break down the starch. IKI is yellow, but turns blue/black in the presence of starch. Once tested with IKI, a yellow color would result indicating no starch. The negative control will vary by student, but should be a substance known to NOT contain amylase. One example of a negative control in this experiment is water because there is no amylase in water. When you add IKI to water, it does not change color. Saliva is included in this experiment because saliva contains amylase. Therefore, the amylase should break down the starch in the starch solution, causing the IKI to turn yellow/brown upon exposure (indicating no starch left after the amylase has broken it down to glucose.) This could also be considered a positive control because we know amylase is present. 2. What is the function of amylase? What does amylase do to starch? (2 pts) Amylase is an enzyme that breaks down starch into simple sugars. It is used for digestive purposes in humans. 3. Which of the foods that you tested contained amylase? Which did not? What experimental evidence supports your claim? (2 pts) Answers will vary by the food products selected. If the IKI was yellow, that meant no starch was present and so amylase has broken it down to sugars. If the IKI turned blue/black, then starch is present, so amylase is not. 4. Saliva does not contain amylase until babies are two months old. How could this affect an infant s digestive requirements? (2 pts) Amylase is required to break down starch in foods like cereal. Babies can t digest cereal or other starch containing foods until their bodies start producing amylase.

3 5. There is another digestive enzyme (other than salivary amylase) that is secreted by the salivary glands. Research to determine what this enzyme is called and identify it. What substrate does it act on? Where in the body does it become activated, and why? (2 pts) The salivary glands offer many different digestive enzymes. One example is lysozyme. Also known as muramidase, this enzyme provides a general antiseptic to kill some of the bacteria which enters the mouth with food. Specifically, it has been found to provide a defense against Gram-positive bacteria such as Bacillus and Streptococcus. It is also found in other body fluids such as tears or milk, and is considered to be part of the innate immune system. NOTE: the question asked for salivary enzymes. Substance Resulting Color Presence of Starch? 6. Digestive enzymes in the gut include proteases, which digest proteins. Why don t these enzymes digest the stomach and small intestine, which are partially composed of protein? (2 pts) The mucosal lining of the digestive tract contains protease inhibitors, preventing the enzymes from digesting the stomach intestines. Furthermore, the coiled structure of the proteins making up the organs hide the cleavage site. Only when proteins are denatured (cooked or in high ph) do their cleavage sites reveal themselves so the enzyme can take effect. Experiment 2: Effect of Temperature on Enzyme Activity Post-Lab Questions (Table - 2 pts) 1. What reaction is being catalyzed in this experiment? (2 pts) 2 H 2 O 2 --> 2 H 2 O + O 2 2. What is the enzyme in this experiment? What is the substrate? (2 pts)

4 Catalase is the enzyme, found in the yeast. (Yeast is a living organism that produces the enzyme catalase. Enzymes are proteins, so connecting with the lecture topic on gene expression--a segment of DNA in the yeast contains the base pairs to make an mrna with the codons that would indicate the correct amino acids to be placed in the right order to make the specific protein, catalase.) The substrate it binds with is hydrogen peroxide. 3. What is the independent variable in this experiment? What is the dependent variable? (2 pts) The independent variable is the different temperature. The dependent variable is enzyme activity which is measured indirectly by the balloon circumference which is dictated by the amount of gas (oxygen) produced by the yeast. 4. How does the temperature affect enzyme function? Use evidence from your data to support your answer. (2 pts) The enzyme function increases as temperature increases. The yeast exposed to the boiling water bath produced the most gas while the yeast exposed to the ice bath/refrigerator produced the least amount of gas. Tube Temperature ( C) Balloon 5. Draw a graph of balloon diameter vs. temperature. What is the correlation? (2 pts) The graph will show the correlation that as temperature increases, balloon circumference increases. This reaction doesn t show it, but most enzymes have a temperature-activity curve that looks like a parabola - activity increases up to a certain temperature, but at higher temperatures the enzyme begins to break down (denature) and activity plateaus and then decreases. 6. Is there a negative control in this experiment? If yes, identify the control. If no, suggest how you could revise the experiment to include a negative control. (2 pts)

5 There is no negative control in this experiment. To include a negative control for this experiment an additional test tube with hydrogen peroxide, but no yeast (enzyme) could be included. If no yeast (enzyme) is present, then no reaction should occur and the balloon should not inflate. 7. In general, how would an increase in substrate alter enzyme activity? Draw a graph to illustrate this relationship. (2 pts) Generally speaking, an increase in substrate alters enzyme activity similar to the way an increase in temperature alters enzyme activity. An increase in substrate will increase enzyme activity until a saturation point has been met; at which point, enzyme activity will plateau and eventually decrease. The graph for this would look similar to the graph provided in Question Design an experiment to determine the optimal temperature for enzyme function, complete with controls. Where would you find the enzymes for this experiment? What substrate would you use? (4 pts) Answers will vary, but the experiment should include all the necessary steps to isolate one variable and include a control(s). You should have indicated what the independent, dependent, and controlled variables, and what you would use as a control. Example: One way would be to use pineapples to produce the enzyme bromelain. The substrate that could be used is the protein in meat that gets broken down by bromelain, which causes the meat to be more tender. A negative control you could use would be a piece of meat that was left untreated. All the meats would be identical cuts and source.