Pearson Mastering Biology: Enzymes - Interactive Lab

When environmental conditions change outside of the optimal range, the enzyme is denatured and no longer catalyzes the reaction. Using the graph, answer the questions below about denaturation of enzymes. In the human body, the enzyme pepsin will denature and not catalyze when the pH of the stomach is above __________. The enzyme trypsin will denature and not catalyze when the pH of the intestine is below __________.

1. 3.5 2. 5.5

Kitchen Items and the Effects of Environmental Conditions You know that the protease (bromelain) in the chef's meat tenderizer will break down the proteins of the blood under optimal conditions. Did the chef commit the crime alone or did the chef have (an) accomplice(s)? The other suspects' fingerprints were found on ice, boiling water, vinegar (an acid), and baking soda (a base). What will happen to the blood when those substances are used with the meat tenderizer? Will the changes in the environmental conditions caused by those products enhance the breakdown of the blood droplets at the crime scene, or denature the protease and stop the breakdown of the blood? What five samples will you test? Match a description of each sample to its number.

1. blood + meat tenderizer 2. blood + meat tenderizer + ice 3. blood + meat tenderizer + boiling water 4. blood + meat tenderizer + vinegar 5. blood + meat tenderizer + baking soda

Experimental Design You identified these five samples to test. As part of the experimental design, you will test each one for the presence of blood to evaluate if the enzyme's function has been affected by the kitchen products (environmental conditions) added by the suspects. Match the samples to the correct sample descriptions based on how the protease reacted with the added kitchen products.

1. blood + protease 2. blood + protease denatured by a low temperature 3. blood + protease denatured by a high temperature 4. blood + protease denatured by a low pH 5. blood + protease denatured by a high pH

Independent and Dependent Variables Review the example provided. Here, the independent variable is the amount of studying and the dependent variable is the grade received on the test. What is the independent variable in your hypothesis? __________ What is the dependent variable in your hypothesis? __________

1. environmental conditions 2. detection of blood

Setting Up the Graph In the Pre-Lab, you identified the independent variable as environmental conditions, and you identified the dependent variable as detection of blood in the sample. Using your independent and dependent variables, begin to create a graph of enzymatic reactions. LABEL FOR X-AXIS __________ LABEL FOR Y-AXIS __________

1. environmental conditions 2. detection of blood

Enzymes Enzymes are protein catalysts often ending with -ase (plural -ases) and named for the molecule with which they react. In today's laboratory, you will use a protease (also called proteinase and proteolytic enzyme). Proteases are produced by all organisms and viruses, including pineapple. What do you think sucrase is? A(n) __________ that breaks down sucrose (sugar). What do you think a protease is? An enzyme that breaks down __________.

1. enzyme 2. proteins

EXPERIMENTAL DESIGN Experimental and Control Groups Now, correlate the sample description to the relationship between substrate (blood) and enzyme (protease) or control. Each relationship illustrates an experimental or control group that will be tested in the experiment. Match the sample and sample descriptions to their correct experimental/control group description.

1. substrate + enzyme 2. substrate + enzyme denatured by low temperature 3. substrate + enzyme denatured by high temperature 4. substrate + enzyme denatured by low pH 5. substrate + enzyme denatured by high pH 6. positive control / correct substrate 7. negative control / wrong substrate 8. enzyme 9. wrong substrate + enzyme

SUMMATIVE QUESTION Kastle-Meyer Test for Blood The following question will be scored. After completing the Kastle-Meyer Test, the cotton swab appears as shown in the image. What can you conclude?

Blood is present in the sample.

Next Steps Your career as a forensic pathologist will take you to many more crime scenes where criminals try to remove blood droplets to cover up their crimes. The following question will be scored. What other successful methods may aid in breaking down blood droplets? What other factors could you add to the experiment to test your hypothesis? Select all that apply.

Decrease the substrate concentration. Increase the protease concentration.

Hypothesis You will now formulate a hypothesis about how the enzyme (protease bromelain in the meat tenderizer) breaks down the substrate (blood) in different environmental conditions. What conditions affect the ability of the enzyme to catalyze the reactions (i.e., what affects the ability of protease to break down blood and therefore affects detection of blood)? Select the best hypothesis.

If the active site of the blood is blocked by an inhibitor, then the blood will not break down. (later question answers only work with this hypothesis)

Role of Inhibitors Some inhibitors are harmful, including poisons that prevent required enzymes from catalyzing reactions. The infamous death cap mushroom, Amanita phalloides, produces poison that inhibits RNA polymerase enzyme from making RNA and leads to cell death. Other inhibitors are beneficial. Consider why your body may want to temporarily stop an enzyme. The following question will be scored. Which of the following is an example of a beneficial inhibitor?

Inhibitor stops the enzyme from catalyzing a reaction of which there is a surplus of the product.

Did any of the results surprise you or contradict your prediction?

It didn't surprise me.

Evaluate Hypothesis Now, consider whether the results of the experiment supported your hypothesis. What inhibitor was tested in your experiment? Did you test an inhibitor?  You selected the hypothesis: If the active site of the blood is blocked by an inhibitor, then the blood will not break down. The following question will be scored. Did the results of your experiment support your hypothesis?

My hypothesis was not supported.

Data Interpretation and Analysis The following question will be scored. What happened to blood when the environmental conditions (temperature or pH) were changed?

No enzymatic reaction.

Data Interpretation and Analysis The following question will be scored. What happened when the wrong substrate (water) was combined with the enzyme?

No enzymatic reaction.

What Would You Expect? Environmental conditions of temperature and pH were changed when you added ice, boiling water, vinegar, or baking soda to the blood + protease enzyme (meat tenderizer); as a result, no enzymatic reaction occurred and blood was detected in those samples. The following question will be scored. What do you expect would happen if the environmental conditions were changed because another kitchen item, salt, was added to the blood and protease?

No enzymatic reaction.

What Would You Expect? What if an inhibitor were added to the blood + protease enzyme (meat tenderizer)? Protease inhibitors are found in many kitchen items, including potatoes, mustard, squash, cereals, legumes, apples, bananas, raisins, cabbage, cucumbers, spinach, and tomatoes. The following question will be scored. What do you expect would happen if a protease inhibitor was added to the blood and protease?

No enzymatic reaction.

Optimal Conditions for Blood Removal As a forensic pathologist, you have had your share of studying blood. A laundry soap company approaches you for your expertise in using enzymes to remove blood.  Although enzymes will denature at icy cold temperatures as you experienced in this lab, proteases work best at cool temperatures. Therefore, you recommend using cool (not icy) water when washing blood from clothing. What else would you recommend? The following question will be scored. Which of the following recommendations would you make to the laundry soap company to create the optimal conditions for blood removal using enzymes? Select all that apply.

Use a protease. Use a neutral pH soap.

EXPERIMENTAL DESIGN Controls In an experiment, a control is the group that experimental groups are compared with to determine the validity of the results. There are both negative controls and positive controls EXAMPLE When testing for the presence of lipids, the negative control is water because it is known that water does not contain lipids, and the positive control is lipid solution because it is known to contain lipids. Each experimental group is compared to the negative and positive controls. If the experimental group is similar to the negative control, then the experimental group is negative for what is being tested. If the experimental group is similar to the positive control, then the experimental group is positive for what is being tested. The following question will be scored. When testing for blood, which of the following is the positive control?

blood

KNOWLEDGE CHECK Enzymatic Reaction The following question will be scored. In which of the following did an enzymatic reaction occur? Select all that apply.

blood + protease cotton swab with clear color of liquids (see image 1)

How Enzymes Work Proteases are enzymes that break down proteins. Sucrase is an enzyme that breaks down sucrose (sugar) into glucose and fructose (sugars).  Other enzymes combine reactants together, such as the enzyme sucrose synthase that combines glucose and fructose to make sucrose. Consider more ways that enzymes can act as catalysts. How do enzymes catalyze biological reactions? Select all that apply.

breaking apart reactants straining bonds binding reactants bringing reactants together enabling alternate pathways with lower energy costs

SUMMATIVE QUESTION Are Enzymes Universal? Of the following substrates (sugar, olive oil, meat, blood, water, and salt), which ones will proteases interact with and catalyze the reactions? Select all that apply.

meat and blood (second and third option)

Making Predictions You will detect the presence or absence of blood in each sample. In which samples do you predict the protease will break down the blood into amino acids (an enzymatic reaction called proteolysis ) and, therefore, blood will not be detected? In which samples do you predict that no catalytic reaction will occur and blood will be detected? Fill in the last column of the table with your predictions.

no yes yes yes yes yes no no no

EXPERIMENTAL DESIGN Negative Control You also add samples of water, meat tenderizer, and water + meat tenderizer. These samples do not contain blood, so they will be used for comparison. The following question will be scored. Which one is your negative control?

water

SUMMATIVE QUESTION Which of the following statements about enzymes are correct? The following question will be scored. Select all that apply.

Each enzyme has a specific substrate with which it can react. Each enzyme can only catalyze a specific reaction. Enzymes are not used up after a reaction; they can be reused over and over for the same reaction. Enzymes only catalyze reactions within a specific range of environmental conditions.

SUMMATIVE QUESTION Identifying Treatments and Controls Correctly place the following samples into the categories of Experimental Groups or Control Groups.

Experimental groups: blood + enzyme denatured by temperature change blood + enzyme denatured by pH change blood + enzyme Control groups: blood water water + enzyme enzyme

Graphs With the environmental conditions, such as temperature or pH, on the x-axis and the detection of blood on the y-axis, which of the following graphs correctly shows enzymatic activity?

Graph 3

Data Interpretation and Analysis The following question will be scored. What happened to blood when combined with the enzyme?

Proteolysis occurred, resulting in no detection of blood.

Enzymes Lower Activation Energy Enzymes lower the activation energy (Ea), the minimum amount of energy that must be spent to initiate a reaction. Let's take a look at two reactions and their activation energy (Ea). The reactants and the products of Reaction 1 and Reaction 2 are the same, but one of the reactions has an enzyme and the other reaction does not. Which reaction occurred with an enzyme and which reaction did not?

Reaction 1: no enzyme Reaction 2: enzyme

A World Without Enzymes Life would not exist without enzymes. Our planet would lack even the most basic bacteria. Why? The following question will be scored. Why can't life exist without enzymes?

Reactions in the body would occur too slowly.

Question Who tried to cover up the crime by removing the blood droplets? Select all that apply.

Suspect 5: The Chef

What Could Go Wrong? Experiments do not always occur as planned. The following is a list of potential scenarios that could have happened in this experiment. The following question will be scored. Which of the following scenarios indicate something went wrong and the experiment needs to be restarted, potentially with new samples? Select all that apply.

The cotton swab of the negative control appears pink. The cotton swab turns pink after the phenolphthalein is added and before hydrogen peroxide is added. The cotton swab of blood looks like this. (see image 1)