Idea Transcript
Biology 101
PCC – Cascade
Name:
Pre-Lab Homework for Lab 5: Enzymes & Diffusion After reading over the lab, answer the following questions. This sheet is due at the beginning of lab! 1. When we discuss the process that your cells use to control reactions, knowing the following terms will be helpful. They can all be found in your text! Catalyst Enzyme: Substrate:
2. What does the suffix "-ase" probably mean when you see at the end of a word in a biology class? (You may need to read the lab to find this – it really is in there!)
3. Draw each of the bags you will make during Exercise # 3. [Hint: There are a total of four!] Be sure to include both internal and external sucrose concentrations. (We have drawn the first for you) % sucrose
% sucrose
Cell A
Cell B
Cell C
Cell D
4. For the bags you have drawn (in question #3) we will be trying to determine which way water is flowing (either into or out of the bag). How will we be able to tell which way water is flowing?
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Biology 101
PCC – Cascade
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Biology 101
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Lab 5: Enzymes & Diffusion GOALS: After successfully completing this lab a student will be able to: •
Show an appreciation for the importance of enzymes in living organisms.
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Investigate factors that control the rate of enzyme-catalyzed reactions.
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Explain how changes in environmental factors can change enzyme-catalyzed reaction rates.
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Demonstrate an understanding of the nature and outcomes of diffusion.
Background: Enzymes are very special types of protein that can cause chemical reactions to speed the rate at which they occur. Because the enzymes are not used up during the reaction an individual enzyme can help hundreds, or even thousands, of reactions to occur before they eventually wear out. So how can enzymes cause reactions to happen more quickly? They have a specially shaped region, called an active site, that binds to the chemicals taking part in a reaction (these chemicals are collectively referred to as the substrate). The structure that is formed is called an enzyme-substrate complex. This complex holds the substrate(s) and helps it (or them) make the transition from one form to another. The basic order of these reactions is often written like this: Enzyme + substrate(s) → Enzyme/substrate complex→ Enzyme + product(s) As you can see from this reaction the enzyme is the same at the beginning and the ending of the reaction but the substrate has now become the product - it’s a chemical reaction! This reaction will occur only if the shape of the active site holds the substrate in just the right orientation. Therefore the shape of the enzyme is the key to its activity. In the experiment that you will perform today you will investigate the activity of an enzyme known as catalase (almost all enzyme names end in the suffix -ase, so any time you see this word ending you can probably assume it is describing an enzyme!). This enzyme speeds up the reaction of hydrogen peroxide (H2O2) into water and oxygen gas and it plays a key role in regulating cellular levels of hydrogen peroxide (this is the same stuff your mom may have poured on your cuts to kill "germs" - it fizzes and bubbles!). The reaction is usually written like this: H2O2 -------------> H2O + ↑O2 catalase Notice how the enzyme name is written under the arrow of the reaction - this shows that an enzyme is used but, as we know, it does not take part in the reaction. We will be investigating the effects of changes in several variables on the ability of this enzyme to speed up this reaction.
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Biology 101
PCC – Cascade
Exercise 1: The effect of enzyme concentration on reaction rate To set up our experiment we will go through the steps of the scientific method, which we can summarize as: Observe → Question → Hypothesize → Test → Conclude Since we are setting up much of this experiment for you we will give you much of the information you will need for this process. OBSERVATION: Many people have made the observation that changing conditions can change reaction rates for enzyme catalyzed reactions. QUESTION: What are some of the conditions that if changed will cause a change in reaction rate? HYPOTHESIS: For this first experiment we will all test the same hypothesis – that changing concentrations of enzymes will cause changes in the rate of a reaction. (Later on, in exercise 2 you will get to investigate another changing condition) EXPERIMENT: The basic experiment will test for the activity of the enzyme catalase under different concentrations of the enzyme. As a source of the enzyme we will use cubes of living potato. (Catalase is found in almost every living cell so when you cut up a potato there is a lot of it around). For this experiment the important variables are: Dependent variable: activity of enzyme - we will measure this by just looking at how fast the bubbles come off the potato. Independent variable: the amount of enzyme added. In this case we will vary the amount of potato added and so change the amount enzyme. Controlled variables: What are some other things that may affect enzyme activity? These are variables you want to make sure are the same from one experiment to the next. After looking over the procedures we will ask you about these again. (question #4) PROCEDURE: 1. Collect from your supply area 5 test tubes, a grease pencil, a test tube rack, the small ruler, and your 10ml graduated cylinder. 2. Label the test tubes 1-5 with a grease pencil.
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Biology 101
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3. Cut the potato so that you have 4 cubes ranging in size from 0.5 cm on a side to 2 cm on a side. (A 1-cm cube is sometimes called 1 cubic centimeter or 1cc and has a volume of 1ml) *please be careful with the very sharp razors* 4. Now set up the tubes as follows: •
Tube 1 - Add a small pinch of sand and 3 ml of water.
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Tube 2 - Add your smallest cube of potato cut into quarters and 3 ml of water.
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Tube 3 – Add your next smallest cube of potato cut into quarters and 3 ml of water.
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Tube 4 – Add your 3rd smallest cube of potato cut into quarters and 3 ml of water.
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Tube 5 – Add your largest cube of potato cut into quarters and 3 ml of water.
Predictions: Now that you know what is in each test tube look over your hypothesis and, based on it, predict the outcome of this experiment. In other words what will happen in each test tube? Fill in the prediction column on the data table. 5. You are now ready to start your experiment. You will estimate the amount of enzyme activity by observing the amount of bubbling coming off of the potato. 6. After waiting about 3 minutes add 4ml of hydrogen peroxide to each of the test tubes and observe the bubbles. Then fill out the following data table using this scale. -
no bubbles
+++
lots of bubbles
+
a few bubbles
++++
oh my gosh the bubbles!
++
moderate bubbles
Tube #
Size of potato cube
Predictions
1 2 3 4 5
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Observed Reaction
Biology 101
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CONCLUSIONS: 1. Do your results agree with your predictions?
2. What does this tell you about the effect of enzyme concentration on enzyme activity? Explain.
3. Why are we making you do test tube 1? (Hmm....something about control variables….?) Explain your answer!
4. What are some of the control variables that you thought of that could change your experimental outcomes?
5. What else could be done to improve this experiment?
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Biology 101
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Exercise 2: The effect of changing environment on reaction rate In this exercise we will investigate either the effects of pH or temperature on our enzyme +
catalyzed reaction. Recall that pH is simply a measure of the number of hydrogen ions (H ) that are floating around in the solution you are measuring. Both pH and temperature are variables that are known to alter some enzymes activity levels. Your job for this exercise is to investigate one of these variables to determine how it affects the activity of the catalase found in potato. After being assigned a variable by your lab instructor take some time to answer the following questions. 1. What variable is your group investigating? 2. For your experiment what are the following: Dependent variable:
Independent variable:
Controlled variables:
3. What do you think will happen to the dependent variable as you alter your independent variable? (This is your prediction.)
4. Why do you think this (your answer to #3) will happen? (This is your hypothesis.)
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Biology 101
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5. Your procedure: You need to write out, in a step by step fashion (write out each step!), the procedure you will use to test your hypothesis (your answer to question #4) by testing to see if your prediction (your answer to question #3) comes true. Step 1:
Step 2:
Step 3: (add more steps as needed…)
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Biology 101
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6. Once you have completed your procedures check them with your instructor and then build a data table in the space below to help you collect your results.
7. Now perform your experiment and record the results in the table you built for question #6. 8. Do your results match your prediction? 9. If they do is your hypothesis proven? If they don’t is your hypothesis disproven? Explain
10. Talk to one of the groups that experimented on the other variable. What hypothesis seems most likely based on their results?
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Biology 101
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11. Briefly summarize what these experiments have shown you about how enzymes work? Be sure to think about what happened and why it happened!
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Biology 101
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EXERCISE 3: Osmosis in a mock cell Diffusion is the basic mechanism that causes the spreading out of materials in either a liquid or a gaseous state. The molecules spread out, moving from high concentration to low spontaneously in a process that doesn't require additional energy. Inside living cells this process takes place in a liquid environment that is organized into compartments by the membranes of the cell. These membranes act as selective barriers, allowing some substances to pass freely through them while blocking the passage of others. Because water molecules are small and have relatively little charge they generally pass freely through a cell's membranes. The direction of this movement is dependent on the concentration of the water on either side of the membrane. The diffusion of water across a membrane is called osmosis and is a very important process for living organisms, as you will see. In this exercise we will build “mock cells” made from dialysis tubing to demonstrate osmosis. The dialysis tubing acts as a stand in for a cells membrane and is selectively permeable allowing small molecules to pass freely while larger molecules are blocked. PROCEDURE: 1. Make 4 "cells" using dialysis tubing by opening the water-soaked tubing and tying one end to form a bag. Fill each bag half full with one of the following: 5% sucrose to Cell A,
distilled water to Cell B,
10% sucrose to Cell C,
25% sucrose to Cell D.
Use a pencil to write the letter A, B, C, D on a piece of paper that is small enough to insert into the bag. Please do not discard the leftover solutions. 2. Loosely fold the end of the bag and squeeze it gently to push out trapped air. Twist the open end of the bag and tie it as you did previously. The bag should be limp after it is tied. 3. Squeeze your bag gently to check for leaks. Leaks will cause you to have odd results. Rinse the bag with tap water and blot the bag with paper towel or KimWipe to remove excess water. 4. Weigh your bag to the nearest 0.1 g and record its weight in the column labeled "0 min/Initial Wt" in Table 1. Place bag A in a beaker of 25% sucrose, and bags B, C and D in a beaker of distilled water. Record the time here: 5. After setting up your experiment take a few moments to make a prediction about what will happen to the weight of each bag and fill out the predicted weight change column of table 1. 6. After 60 minutes remove the bag, gently blot excess water, and weigh it to the nearest 0.1 g. Record the weight of the bag in Table 1. Also record the change in weight of the bag. Indicate weight gain with a plus (+) and weight loss with a minus (-). 11
Biology 101
PCC – Cascade
7. In this experiment we are examining the effects of concentration on weight gain/loss to better understand osmosis. What is the dependent variable in this experiment? the independent? Table 1: Weight change over time: Bag
predicted weight change
wt. at beginning of experiment (g)
wt. at end of experiment (g)
observed weight change
A B C D
8. In this experiment we expected the weight of the bag to change. Is this change due to the movement of sugars or the movement of water? Explain your answer.
9. Explain the weight change in the bags based on the principles of osmosis (If the bags didn't change try to explain that!). Be sure to refer to a water gradient and to the net movement of water. Bag A
Bag B
Bag C
Bag D
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