JoVE Lab Manual
Lab Bio
Lab Bio
A subscription to JoVE is required to view this content.
Enzyme Activity
Learning Objectives
At the end of this lab, students should know...
What are enzymes?
Enzymes are biological catalysts that increase the speed and efficiency of biochemical reactions. Most enzymes are proteins but certain ribonucleic acid molecules also have catalytic properties.
How do enzymes catalyze reactions?
Enzymes catalyze reactions by lowering the activation energy of a reaction.
What are catabolic and anabolic reactions?
Most enzymatic reactions can be categorized as either catabolic or anabolic, which have opposite functions. Enzymes that mediate catabolic reactions break down larger substances into multiple products. Anabolic enzymes combine multiple substances into a single product.
How do changes in conditions affect enzyme activity?
Changes in conditions in an enzyme’s environment can cause the enzyme to not initially fold properly, to change shape after initial folding, or can change the chemical properties of the active site, all of which can prevent the enzyme from effectively binding with a substrate or catalyzing a reaction.
Why is it important to study enzymes and their reaction rates?
Enzymes are essential for most biochemical reactions in organisms. Understanding human enzyme functions and kinetics helps with development of therapies to modify functions of defective enzymes of some individuals. Also, enzyme inhibitors can be used as therapeutics against microorganisms or cancer. Moreover, protein engineering can develop enzymes with various functions, such as bioremediation.
List of Materials
-
Test tubes200
-
Test tube rack10
-
Dropper5
-
Timer5
-
pH Chart5
-
Label tape5
-
Marker5
-
Camera/phone with camera5
-
Parafilm/sealing film1 per station
-
Hot plate1 per station
-
Ice cube (1/2 tray)1 per station
-
Thermometer1 per station
-
pH meter/pH strips1 for whole lab
-
Knife1 for whole lab
-
Blender1 for whole lab
-
Cheese cloth (0.5 mtrs)1 for whole lab
-
Coffee filter1 for whole lab
-
0.1 M sodium phosphate diobasic soln (250 mL)Dependent on the lab size
-
0.1 M sodium phosphate monobasic (250 mL)Dependent on the lab size
-
Turnip (1-2)Dependent on the lab size
-
Hydrogen Peroxide (3%) (15 mL)Dependent on the lab size
-
Guaiacol (concentrtion soln, 1-3mL)Dependent on the lab size
-
Isopropyl rubbing alchohol (100mL)Dependent on the lab size
-
pH buffer capsules/pH (pH range- 3-8)Dependent on the lab size
Lab Prep
- Investigating the Effect of pH and Temperature on Peroxidase Activity
- To make the extraction buffer for the peroxidase enzyme, mix equal volumes of 0.1 M sodium phosphate monobasic and 0.1 molar sodium phosphate dibasic solutions to achieve a final volume of 500 mL.
- Test the resulting solution using a pH meter. The mixture should be between a pH of 6.8 and 7.2.
- To extract the turnip peroxidase, use a knife to remove the outer layer of a turnip and then cut the turnip into approximately two cm cubes.
- Place 30 g of turnip cubes into a blender with 300 mL extraction buffer, and blend the turnip cubes at high speed three times for about 15 s each time.
- Filter the homogenized mixture through three layers of cheesecloth, then strain the resulting solution through one coffee filter.
- Store the filtrate in a 500 mL bottle, and immediately place the bottle in the refrigerator.
- Next, to make the enzyme substrate solutions, first add 15 mL 3% H2O2 to 435 mL distilled water to dilute the H2O2 to a 0.1% concentration.
- To prepare the color-changing reactant, dissolve 0.2 mL concentrated guaiacol in 100 mL of isopropyl rubbing alcohol, and store this guaiacol solution in the refrigerator.
- Then, to make solutions for the different pH conditions, add 100 mL distilled water to six 250 mL beakers, and place the buffer capsule contents with pH of 3, 4, 5, 6, 7, and 8 into separate beakers.
