Enzyme Lab

Biology Name: Abbie, Clare, Hope, and Naomi

Peroxidase Enzyme Lab

Peroxidase Enzyme Lab

Question

How do abiotic or biotic factors influence the rates of enzymatic reactions (chemical reactions that are assisted by enzymes)?

Background

Enzymes speed up chemical reactions by lowering activation energy (that is, the energy needed for a reaction to begin). In every chemical reaction, the starting materials (the substrate(s) in the case of enzymes) can take many different paths to forming products. For each path, there is an intermediate or transitional product between reactants and final products. The energy needed to start a reaction is the energy required to form that transitional product. Enzymes make it easier for substrates to reach that transitional state. The easier it is to reach that state, the less energy the reaction needs. Enzymes are biological catalysts. They are large protein molecules, folded so that they have very specifically shaped substrate binding sites. These binding sites make substrates go into the transition state. To catalyze the reaction, several regions of the binding site must be precisely positioned around the substrate molecules. Any change in the shape of the overall folded enzyme molecule can change the shape of the binding site. The optimum reaction conditions are different for each enzyme. The correct
environmental conditions, proper substrates, and, often, particular cofactors associated with an enzyme are needed. In some instances, the optimum conditions can be deduced fairly accurately based on the following: 
The organism from which the enzyme is derived
The part of the organism in which the enzyme functions 
The environmental conditions in which that organism lives

Take the example of lactase, an enzyme that catabolizes (breaks down) the disaccharide sugar lactose into the two monosaccharides, glucose and galactose. In humans, lactase is found mostly in the small intestine, where the pH is around 7. It would be reasonable to hypothesize that human lactase is optimally active at pH 7 and at 37°C (normal human core body temperature in degrees celsius). Free-living decomposer fungi in soil also produce lactase. However, soil pH usually is between 5 and 6.5. As could be predicted, the purified enzyme from a common soil fungus has a pH optimum of 5.5. The main enzyme for this lab, peroxidase, is found in many different forms, with optimum pHs ranging from 4 to 11 depending on the source and optimum temperatures varying from 10 to 70°C.

Purpose

In this experiment you will investigate the effect of environmental factors on the enzyme hydrogen peroxidase. This enzyme is found in all aerobic (using oxygen) cells and functions to decompose hydrogen peroxide into O2(g) and H2O. The specific environmental factors you will test (as a class) are temperature, pH, substrate concentration, and enzyme concentration. Your team will select one of these factors (variables) to test and report on.

Materials

• 5 to 10 grams of freshly picked ripgut bromegrass (Bromus diandrus) blades (about 1 handful)
• Digital balance (scale)
• Mortar and pestle
• Distilled water
• 3 100-liter glass or plastic beakers
• 1 mL or 5 mL syringe
• Hydrogen peroxide
• 1 Paper towel square (for filtration)
• Glass test tubes
• Test tube rack or holder
• Small plastic ruler
• Safety glasses

Depending on which environmental factor you choose to investigate, some of the following items will be needed for your experiment:

• Acid solutions with pH values between 2 and 6
• Alkaline solutions with a pH between 8 and 12
• pH test strips
• Ice
• Large plastic beaker (for ice bath)
• Hot water
• Large plastic beaker (for hot water bath)
• Thermometers

Procedure

This part will be determined by the students conducting the experiment. For an overview of the general scientific experimentation and research process, see the flow diagram below. Use the worksheet that follows to write out in detail the hypothesis you are seeking to test, the materials you will use, and the steps you will follow to conduct your experiment.

Enzyme Lab Worksheet

Hypothesis: We predict that increasing the amount of enzyme concentration will speed up the reaction process, because there are more proteins acting as catalysts to lower the activation energy for the reactions.

Independent Variable: Enzyme concentration (mL)

Dependent Variable: Rate of reaction (seconds)

Controlled Variables: Amount of grass (substrate concentration), amount of water, temperature, pH

Justification of hypothesis:

We chose this hypothesis because if you increase the enzyme concentration then it’s like adding more workers to work on a project, which would be more efficient, and speed up reaction time.

Materials (Your Team’s Experiment):

• 5 to 10 grams of freshly picked ripgut bromegrass (Bromus diandrus) blades (about 1 handful)
• Mortar and pestle
• Distilled water
• 3 100-liter glass or plastic beakers
• 1 mL or 5 mL syringe
• Hydrogen peroxide
• 1 Paper towel square (for filtration)
• Glass test tubes
• Test tube rack or holder
• Safety glasses

Procedure:

1. Use the mortar and pestle to mush grass
2. Put grass and water mixture into test tube
3. Add different amounts of hydrogen peroxide to test tube
4. Record the height of the solution at different time intervals

Summary: Once the chemicals are released from the grass, add more hydrogen peroxide to increase the enzyme concentration.

Detailed Steps:

Crush 10 blades of grass in the mortar with 2 mL of water to make grass juice.

Pour the grass juice into a test tube (2 ml of mixture), using a paper towel as a filtration device

Add 1 ml of hydrogen peroxide (the enzyme) into the test tube containing the grass juice. As you pour the hydrogen peroxide, start a timer.


Record the height of the new solution during its reaction process every 10 seconds for 1 minute.


Repeat steps 1-4 with 2 mL of hydrogen peroxide.


Repeat steps 1-4 with 3 mL of hydrogen peroxide.


Interpret and graph the results

Data and Results:

Time (seconds)	Trial 1: 1 mL Hydrogen Peroxide	Trial 2: 2 mL Hydrogen Peroxide	Trial 3: 3 mL Hydrogen Peroxide
0	2 mL	2 mL	2 mL
10	4.5 mL	5.5 mL	6 mL
20	5 mL	6 mL	6.7 mL
30	5.7 mL	7 mL	7 mL
40	6.5 mL	8 mL	8 mL
50	7 mL	9 mL	9 mL
60	7.5 mL	9.5 mL	10 mL

Graph:

Conclusion:


In the end, our hypothesis was correct. There was more foam faster when we added more hydrogen peroxide. This happens because increasing the enzyme concentration speeds up the reaction process and affects substrates faster. If we were to continue our experiment, we would keep adding more and more enzyme concentration to find the amount that gives the fastest reaction time.