Experimental Procedure
Making a Catalase Enzyme Solution
In this part of the project, you will make a catalase enzyme solution from a potato. You will also test the enzyme to make sure it is active at room temperature, and that its activity is not too high or too low at room temperature. The catalase solution should show a level of activity at room temperature that will give you enough range to see increases or decreases in the activity when you test it at other temperatures.
Note: It is best to make your catalase solution right before you plan to use it. The enzymes in the solution will begin to denature (or stop working) within a couple of hours, so you do not want to make this solution in advance.
1. Put the distilled water in the refrigerator to cool it down while you get ready to use it. 2. Cut a coffee filter into small squares.
a. Make each square 3 x 3 centimeters (cm). b. You will need at least 18 squares total.
3. Fill the ice chest about 5 cm deep with ice. Close the ice chest. 4. Crush about eight ice cubes.
a. Crush the ice in a 1 gallon sealable plastic bag using a hammer or wooden mallet so that the largest pieces are no longer than 2 cm.
b. You will want to do this on a hard surface, such as concrete, and be careful not to damage it. c. When you have finished crushing the ice cubes, put the bag in the ice chest with the other ice. 5. Carefully peel a raw potato, cut it into small pieces, and weigh out 50 grams (g), as shown in Figure 2
below.
a. Cut the potato so that no pieces are larger than 3 cm.
b. To keep the potato pieces clean and uncontaminated, weigh them on a piece of wax paper. Make sure to zero out the scale with the wax paper on it before weighing out the 50 g of potato pieces on the wax paper.
Science Buddies Kit: Use the plastic weigh boat included in the kit instead of a piece of
Figure 2.On a small piece of wax paper (or weigh boat) on the scale, weigh out 50 g of small potato pieces. Be sure to zero the scale after placing the wax paper or weigh boat on the scale but before adding the potatoes.
6. Place the potato pieces, 60 milliliters (mL) (¼ cup) of cold distilled water, and approximately 30 mL (2 tablespoons [tbsp.]) of crushed ice in a blender.
7. Blend the potato pieces, distilled water, and crushed ice for 20 seconds at high speed.
8. If the blender jar is small enough to fit, put it inside of the small ice chest, surrounded by ice. This will keep the iced solution, which is the catalase solution, chilled.
a. If the blender jar does not fit inside the ice chest, pour the blended, iced catalase solution into a large jar or cup and put it inside of the small ice chest, surrounded by ice. Alternatively, if you are ready, you can immediately proceed to step 9, but keep the catalase solution off of the ice for as little as possible.
b. Rearrange the ice so that it completely surrounds the catalase solution container (but make sure no ice falls into the solution), as shown in Figure 3 below. Add more ice to the ice chest if needed.
c. From this point on, leave the catalase solution in the ice in the small ice chest as much as possible.
Figure 3. Once you have prepared your catalase solution, keep it stored in the ice chest on ice, with ice arranged so that it is surrounding the jar.
9. Over another large jar or cup (which should be able to stably hold at least 400 mL, or about 1 ¾ cups), place a coffee filter and carefully pour your catalase solution into the coffee filter, as shown in Figure 4 below. Hold the coffee filter over the new jar to make sure that it does not sink down into the jar. To give it more support, you could place the coffee filter in a funnel and set it so that it drains into the new jar.
o Science Buddies Kit: The kit includes a funnel for you to use during this step.
b. Be careful not to pour too much catalase solution into the coffee filter at once or it might become
too heavy or tear.
c. If you let the catalase solution filter for a few minutes and a lot of liquid is still trapped in the
coffee filter, get a new jar or cup and carefully and gently squeeze the liquid through the filter over the new jar.
i. You will need to use a new jar in case the filter tears, releasing unfiltered pulp into the jar.
ii. When you have squeezed out most of the remaining liquid, pour the liquid from this new jar into the jar with the original filtered catalase solution. Pouring the liquid slowly can trap any unfiltered pulp (that may have accidentally spilled out) at the bottom of the jar.
Figure 4. Filter the catalase solution through a coffee filter and into a new large jar or cup. Be sure to hold on to the coffee filter as you filter the catalase solution. If you have a funnel, place the coffee filter inside the funnel and position the funnel so that the solution drains into the jar.
10. Add 175 mL (3/4 cup) cold distilled water to the filtered catalase solution. Mix well.
11. Put the catalase solution back on ice. Add more ice to the ice chest so that the catalase solution in the jar is still surrounded by ice.
12. Test your catalase solution to see how well it works at room temperature.
a. If the catalase enzyme works too quickly at room temperature, it will be hard to measure its activity as it increases at other temperatures. If the catalase works too slowly, then it will take a long time to measure its activity as it decreases at other temperatures.
b. In a clean baby 4 oz. food jar, add 40 mL (2 ¾ tbsp.) of hydrogen peroxide.
i. Make sure the hydrogen peroxide is at room temperature for at least an hour before using it for this project.
c. Have a stopwatch or timer ready.
d. Using forceps or tweezers, immerse a 3 x 3 cm coffee filter square into the catalase solution and hold it immersed for five seconds.
e. Remove the coffee filter square and drain off any excess catalase solution by touching an edge of the filter square to the inside of the catalase solution jar.
f. Lower the coffee filter square so that it is just above the surface of the hydrogen peroxide in the baby food jar, and then drop the coffee filter square.
i. Releasing the coffee filter square from right above the surface of the hydrogen peroxide will help prevent the coffee filter square from sticking to the sides of the jar.
the surface again. It is easiest to observe the filter square's movements from the side, instead of from above.
i. Remove and dispose of the filter square, pour the hydrogen peroxide out of the jar, and rinse the jar with water.
Figure 5. When the coffee filter square is soaked in the catalase solution and then dropped into the baby food jar with the hydrogen peroxide, it should initially sink (left) and then rise back to the surface of the hydrogen peroxide (right).
h. Ideally the coffee filter square should take about 20 seconds to rise at room temperature. If it takes less than 15 seconds or more than 25 seconds to rise, you will need to modify your catalase solution so that it takes between 15 to 25 seconds for the coffee filter square to rise.
i. If it takes less than 15 seconds to rise, add 30 mL (2 tbsp.) of cold distilled water to your catalase solution, stir, and repeat steps 12 b to h. You may need to do this multiple times, adding cold distilled water until the filter square takes more than 15 seconds to rise. ii. If it takes more than 25 seconds to rise, prepare and test the catalase solution again
(repeating steps 5 to 12, as well as step 4 if more crushed ice is needed or it has melted) but skip step 10 so that the catalase solution is more concentrated.
iii. If you need to retest your catalase solution, make sure to use new hydrogen peroxide in step 12b each time.
iv. What do you think this says about how the concentration of an enzyme affects its activity?
13. When you have finished testing your catalase solution, add more ice to the ice chest so that the catalase solution in the jar is still surrounded by ice.
Testing The Effect of Temperature on Enzyme Activity
After making the catalase enzyme solution and making sure that it works well at room temperature, you will investigate its activity at a range of different temperatures. The temperatures you will be testing are
degrees C, and 40 degrees C. As before, you will soak a coffee filter square in the catalase solution and then time how long it takes to sink and rise in a jar of hydrogen peroxide. Make a table in your lab notebook to easily record your data. In addition, make sure you clean the baby food jars in between each test. You do not want the catalase left over from a previous test to influence the results of the next test. Read all of the instructions before you start, so that you can decide which will be the most efficient testing method for you.
1. First test the enzyme activity at approximately -18 degrees C.
a. Fill the cleaned baby food jar with 40 mL (2 ¾ tbsp.) of hydrogen peroxide. b. Put the thermometer in the freezer.
c. Put the jar with hydrogen peroxide in the freezer. Check the time or start a stopwatch or timer. d. After the jar has been in the freezer for 20 minutes, test the enzyme activity just as you did in the
"Making a Catalase Enzyme Solution" section in steps 12 c to g.
e. How long did it take the coffee filter square to sink and rise again? Write the time (in seconds) in the table in your lab notebook.
f. Check the thermometer in the freezer and record the exact temperature (in Celsius) in your table. The temperature in your freezer may not be exactly -18 degrees C.
2. Test the enzyme activity at approximately 4 degrees C.
a. Repeat step 1, but this time put the jar and thermometer in the refrigerator instead of the freezer (again for 20 minutes).
b. In your lab notebook, write the time it took for the coffee filter square to sink and rise again, and the exact temperature in the refrigerator.
3. Test the enzyme activity at approximately 20 degrees C.
a. Repeat step 1, but this time keep the jar and thermometer out at room temperature.
b. If the jar and thermometer were already at room temperature, you do not have to wait 20 minutes before testing the enzyme activity and checking the temperature.
c. In your lab notebook, write the time it took for the coffee filter square to sink and rise again, and the exact temperature of the room. It may not be exactly 20 degrees C.
4. Test the enzyme activity at approximately 30 degrees C.
a. To do this you will be cooling down water from near-boiling state (well over 30 degrees C). b. Fill the cleaned baby food jar with 40 mL (2 ¾ tbsp.) of hydrogen peroxide.
c. Fill a cooking pot with water to a depth of about 4 cm.
d. Heat the pot of water on a stove or hot plate until the water just barely boils. Then immediately take the pot off the burner to let it cool.
e. Put the thermometer in the pot of water. When the water reaches approximately 40 degrees C, carefully put the jar with the hydrogen peroxide in the pot of water.
i. Remove some water from the pot if the water level is so high that water might flow into the jar or if the jar floats, even a little.
f. When the water reaches 30 degrees C, test the enzyme activity as you did in the "Making a Catalase Enzyme Solution" section in steps 12 c to g.
g. How long did it take the coffee filter square to sink and rise again? Record this in the table in your lab notebook, along with the exact temperature of the water.
5. Test the enzyme activity at approximately 40 degrees C.
a. To do this you will be cooling down water from near-boiling state (well over 40 degrees C). b. Repeat step 4 with the following changes:
i. On step 4d, when the water reaches 60 degrees C, carefully put the jar with the hydrogen peroxide in the pot of water.
ii. On step 4e, test the enzyme activity when the water reaches 40 degrees C (not 30 degrees C).
iii. In your lab notebook, record the time it took for the coffee filter square to sink and rise again, along with the exact temperature of the water.
a. When performing science experiments, it is important to perform the experiment at least three times to make sure that your results are repeatable and reproducible.
b. If you have multiple clean baby food jars, you can test multiple temperatures at the same time; however, testing multiple temperatures at the same time can get complicated.
c. Note: It is important to test the enzyme activity at the various temperatures as close together in time as possible. If the tests cannot all be done in two to three hours, you will need to prepare and use a new catalase solution, because the original catalase solution may not be good anymore. After three hours you may notice a significant decrease in the enzyme's activity. 7. For each temperature, calculate the average time it took for the coffee filter paper to sink and rise. 8. Graph your results, plotting the temperature (in Celsius) on the x-axis and the time to resurface (in
seconds) on the y-axis.
9. At which temperature did the coffee filter squares take the most time to resurface? At which temperature did they take the least? What does this indicate about the enzyme's activity?
