Name___________________________ AP Bio Lab: Enzymes
Introduction
Enzymes have been found to accelerate chemical reactions by lowering activation energy. The chemical on which an
enzyme actually operates is referred to as a substrate. Substrate concentration, enzyme concentration, pH,
temperature, and the presence of an inhibitor will all affect the performance of an enzyme on a chemical reaction. In
this lab, an enzyme referred to as "turnip peroxidase" will be used to convert a clear solution to a colored solution. An
instrument known as the "Spec 20" will be used to measure the rate of the reaction by measuring the increase in color
as it appears during the reaction. The following equation represents the reaction:
H2 O2
hydrogen peroxide
(clear)
+
C 7H 8O 2
--------------->
(C7H8O2)4
quaiacol
(clear)
enzyme
peroxidase
tetraguaiacol
(orange)
+
H2 O
water
(clear)
A colorimeter measures the amount of absorbance of a solution in a test tube. Inside the instrument is a light that is
allowed to shine through an inserted test tube. An absorbance meter measures how much light has been blocked by
the test tube sample. The amount of colored substance in the test tube is directly proportional to the absorbance read
by the colorimeter. In other words, the greater the concentration, the higher the absorbance.
How to Use the Colorimeter:
1. The colorimeter must be plugged in (to the Go-Link! and Go-Link! plugged into the USB port of a laptop) warmed
up for at least 5 mins and the wavelength set to 470 nm.
2. Find the "Absorb-Conc Colorimeter.cmbl" file on your laptop. It is in: Applications -> Lobber Pro 3 -> Experiments > Probes & Sensors -> Colorimeter -> Absorb-Conc Colorimeter.cmbl. Open the file.
3. The machine must be “blanked” by inserting a square cuvette of solvent for comparison to a test sample. The
blank cuvette serves as a control. Turn the right hand knob to adjust to read zero absorbance.
4. Remove the blank cuvette, insert the sample cuvette, and read its absorbance.
5. The colorimeter must be "blanked" before each sample cuvette is inserted to be sure that the data collected is
accurate.
Reagents
The turnip extract (source of peroxidase) was prepared by blending 2 g of turnip in 200 mL of distilled water. The
solution concentration was adjusted so that its spec 20 absorbance would fall in the range of 0.1 to 0.2. The hydrogen
peroxide solution was prepared at 3%. The guaiacol solution was prepared at approximately 3%.
General Information
1. In this lab you will first carry out what is referred to as a “baseline” reaction. This reaction will be used as a control
to which 6 other reactions will be compared. In the other 6 reactions, you will will vary the enzyme concentration
(double and half), substrate concentration (double and half), and temperature (up 10°C and down 10°C).
2. You will need to obtain 3 larger test tubes and 2 square cuvettes. Label the cuvettes: B (blank), and R (reaction).
Label the larger test tubes: 1, 2, and 3.
3. You will need to obtain 2 (or 3 if available) 10 ml graduated cylinders. Rinse them thoroughly with distilled water.
They will be used to measure the reagents. Reagents are available in dropper bottles. Measure reagents by
dropping them into the graduated cylinder with the dropper bottles until the desired volume is reached.
4. The graduated cylinders are needed for measuring only. Once the reagents are measured, they are to be added
to the appropriate test tube. Add all of the reagents together in the graduated cylinder before pouring the
graduated cylinder contents into the appropriate test tube.
5. You then need to “blank” the spec 20 BEFORE EACH REACTION by pouring the contents of test tube #1 into the
“B” cuvette (3/4 full), inserting the “B” cuvette into the spec 20, and adjusting it’s absorbance to 0.
6. Have a watch ready. When ready, start the time and pour the contents of tubes #2 and #3 together (back and
forth) several times. Then pour the mixed contents into cuvette "R" (to 3/4 full) and insert it into the spec 20
machine. At 20 seconds, obtain your first absorbance reading, and read the absorbance every 20 seconds for 3
minutes. Discard the extra reagent in the mixing tubes that does not fit into the square cuvette.
7. You can save cuvette 1 to serve as a blank for reactions 4, 5, 6, and 7. You will not have to
Name___________________________ AP Bio Lab: Enzymes
Procedure
1. For each of the following reactions, mix the reagents as instructed.
2. After mixing, please perform the appropriate lab procedure from the "General Information" section.
3. Be sure to rinse all test tubes and graduated cylinders thoroughly after each reaction.
4. Note: for each reaction, Tube 1 = blank, Tube 2 = reactants, Tube 3 = enzyme.
5. In Reaction 1, count the drops of quaiacol that are required to add 0.1 ml guaiacol to the graduated cylinder. After
that, always add the same number of guaiacol drops - do not measure 0.1 ml guaiacol.
6.
Reaction 1: Base line
1. Tube #1: mix 8.9 mL of distilled water, 1.0 mL of turnip extract, and 0.1 mL guaiacol.
a. (save blank for reactions 4, 5, 6, 7)
2. Tube #2. mix 4.7 mL of distilled water, 0.2 mL of hydrogen peroxide, and 0.1 mL of guaiacol.
3. Tube #3: mix 4.0 mL of distilled water and 1.0 mL of turnip extract.
Reaction 2: Effect of Doubling the Enzyme concentration
1. Tube #1: mix 7.9 mL of distilled water, 2.0 mL of turnip extract , and 0.1 mL guaiacol.
2. Tube #2: mix 4.7 mL of distilled water, 0.2 mL of hydrogen peroxide, and 0.1 mL guaiacol.
3. Tube #3: mix 3.0 mL of distilled water and 2.0 mL of turnip extract.
Reaction 3: Effect of Halving the Enzyme concentration
4. Tube #1: mix 9.4 mL of distilled water, 0.5 mL of turnip extract, and 0.1 mL guaiacol.
5. Tube #2: mix 4.7 mL of distilled water, 0.2 mL of hydrogen peroxide, and 0.1 mL guaiacol.
6. Tube #3: mix 4.5 mL of distilled water and 0.5 mL of turnip extract.
Reaction 4: Effect of Doubling a Substrate concentration
7. Tube #1: mix 8.9 mL of distilled water, 1.0 mL of turnip extract, and 0.1 mL guaiacol. (already mixed - Rx 1)
8. Tube #2. mix 4.5 mL of distilled water, 0.4 mL of hydrogen peroxide, and 0.1 mL of guaiacol.
9. Tube #3: Mix 4.0 mL of distilled water and 1.0 mL of turnip extract.
Reaction 5: Effect of Halving a Substrate concentration
10. Tube #1: mix 8.9 mL of distilled water, 1.0 mL of turnip extract, and 0.1 mL guaiacol. (already mixed - Rx 1)
11. Tube #2. mix 4.8 mL of distilled water, 0.1 mL of hydrogen peroxide, and 0.1 mL of guaiacol.
12. Tube #3: mix 4.0 mL of distilled water and 1.0 mL of turnip extract.
Reaction 6: Effect of Increased Temperature
13. Tube #1: mix 8.9 mL of distilled water, 1.0 mL of turnip extract, and 0.1 mL guaiacol. (already mixed - Rx 1)
14. Tube #2. mix 4.7 mL of distilled water, 0.2 mL of hydrogen peroxide, and 0.1 mL of guaiacol.
15. Tube #3: mix 4.0 mL of distilled water and 1.0 mL of turnip extract.
16. Heat up the test tubes (#2 and #3) in the warm water bath prior to mixing! Record the temperature from the
thermometer in the warm water bath!
Reaction 7: Effect of Decreased Temperature
17. Tube #1: mix 8.9 mL of distilled water, 1.0 mL of turnip extract, and 0.1 mL guaiacol. (already mixed - Rx 1)
18. Tube #2. mix 4.7 mL of distilled water, 0.2 mL of hydrogen peroxide, and 0.1 mL of guaiacol.
19. Tube #3: mix 4.0 mL of distilled water and 1.0 mL of turnip extract.
20. NOTE: Cool down the test tubes (#2 and #3) in the cool water bath prior to mixing! Record the temperature from
the thermometer in the water bath!
Post Lab - writeup
21. For this lab, staple your data table into your lab book at the start of lab 2 (Enzyme Lab).
22. Put all of your data into the data tables and graph each of the sets of data obtained, absorbance vs. time, on one
large graph, or more than one graph as long as it is clear. You will need to draw smooth curves through the
points. DO NOT JUST CONNECT THE DOTS! Be sure to color-code the different curves, and show a legend in
the margin to indicate the meanings of the colors.
23. Fill out the entire lab 2 in your lab manual. You will need to adapt this lab to your lab manual.
Name___________________________ AP Bio Lab: Enzymes
Data Table - Absorbance ( 2-log[% trans] )
Rx 1
Rx2
20s
40s
60s
80s
100s
120s
140s
160s
180s
Rx3
Rx4
Rx5
Rx6
Rx7
Name___________________________ AP Bio Lab: Enzymes