Enzyme Lab Background and Overview

Question

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

Some abiotic factors that influence the rates of enzymatic reactions are pH levels that alter the strength of hydrogen bonds’ polarity, temperature that changes the speed of the enzyme and substrate and enzyme concentration which gives more possibilities to have a reaction.

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 cabbage (brassica oleracea) blades (about 1 handful)
• Digital balance (scale)
• 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
• Acid solutions with pH values between 2 and 6
• Alkaline solutions with a pH between 8 and 12
• pH test strips

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

• Ice
• Large plastic beaker (for ice bath)
• Hot water
• Large plastic beaker (for hot water bath)
• Thermometers

Procedure

1. First, we are going to take three test tubes, and put 1 mL of grass extract, mL of hydrogen peroxide, and 1 mL of water mixed in each of them. 5 minutes later, we will check the froth.     
2. Next, we will take five test tubes and put 1 mL of hydrogen peroxide in each, 1 mL of grass extract in each, and finally, 1 mL of 3pH buffer solution in the first tube.
3. We then will record the amount of millimeters of the froth grown in that solution with the 3pH buffer solution.
4. Next, after recording the data of the 3 pH buffer solution, with the second test tube, we will place 1mL of 5pH buffer in the second tube.
5. After that, we will record the amount of froth grown, in millimeters, every minute up to five minutes for the 5 pH buffer solution.
6. Then, after recording the data of the 5 pH buffer solution, with the third test tube, we will place 1mL of 7pH buffer in the third tube.
7. After that, we will record the amount of froth grown, in millimeters, every minute up to five minutes for the 7 pH buffer solution.
8. Then, after recording the data of the 7 pH buffer solution, with the fourth test tube, we will place 1mL of 8pH buffer in the fourth tube.
9. After that, we will record the amount of froth grown, in millimeters, every minute up to five minutes for the 8 pH buffer solution.
10. Then, after recording the data of the 7 pH buffer solution, with the fifth test tube, we will place 1mL of 9pH buffer in the fifth tube.
11. After that, we will record the amount of froth grown, in millimeters, every minute up to five minutes for the 9 pH buffer solution.
12. Finally, we will create a chart of all of our data, and observations.

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: If there are different visual reactions with hydrogen peroxide, grass extract and multiple pH solutions, then enzymatic reactions can be dependent upon the pH level.

Independent Variable: Varying pH solutions, either alkaline or acidic

Dependent Variable: Slightly acidic solutions will create a higher reaction rate

Controlled Variables: The pH solution tests

Justification of hypothesis: Very high or very low pH levels will most likely result in a change of the hydrogen bonds’ charge, which will change the polarity, thus breaking the bonds. However, completely neutral solutions will most likely not be able to strengthen or break the bonds, thus seeing no change.

Materials (Your Team’s Experiment):

• Hydrogen peroxide

• Alkaline solutions

• Acidic solutions

• Neutral solutions

• Grass extract

• Dropper

• Test tubes

• PH test strips

• Paper towels

• Mortar and pestle

Procedure:

Summary: In this experiment, our main goal is to test the pH of the enzymes (hydrogen peroxide) on our plant and to see if the reaction rates of the enzymes increase. We took 9 test tubes and tested hydrogen peroxide, grass extract, with 5 different pH solutions, and one with water. We then recorded all of our data in 1 minute intervals, and stopped at five minutes. The largest rate of reaction was the 9 pH solution.

Detailed Steps:

1. First, we took three test tubes, and put 1 mL of grass extract, mL of hydrogen peroxide, and 1 mL of water mixed in each of them. 5 minutes later, check the froth and it will end up to be about 14 mL       
2. Next, we took five test tubes and put 1 mL of hydrogen peroxide in each, 1 mL of grass extract in each, and finally, 1 mL of 3pH buffer solution in the first tube.
3. We then recorded the amount of millimeters of the froth grown in that solution with the 3pH buffer solution.
4. Next, after recording the data of the 3 pH buffer solution, with the second test tube, we placed 1mL of 5pH buffer in the second tube,
5. After that, we recorded the amount of froth grown, in millimeters, every minute up to five minutes for the 5 pH buffer solution.
6. Then, after recording the data of the 5 pH buffer solution, with the third test tube, we placed 1mL of 7pH buffer in the third tube,
7. After that, we recorded the amount of froth grown, in millimeters, every minute up to five minutes for the 7 pH buffer solution.
8. Then, after recording the data of the 7 pH buffer solution, with the fourth test tube, we placed 1mL of 8pH buffer in the fourth tube,
9. After that, we recorded the amount of froth grown, in millimeters, every minute up to five minutes for the 8 pH buffer solution.
10. Then, after recording the data of the 7 pH buffer solution, with the fifth test tube, we placed 1mL of 9pH buffer in the fifth tube,
11. After that, we recorded the amount of froth grown, in millimeters, every minute up to five minutes for the 9 pH buffer solution.
12. Finally, we created a chart showing all of the buffers and solutions, and in one minute intervals, each amount of froth from each one in millimeters was recorded.

Data and Results:

(This section should include at least one graph!)

Graph Showing the Froth Growth for Each Solution.

For Graph: Light Green = Water solution, Purple = 3pH, Orange = 5pH, Red = 7pH,

Blue = 8 pH, and Dark Green = 9pH

Data/Results of the Growth of the Froth in Each Solution                                                                     

Control - 10ml water, 10 ml grass extract, 10 ml 3% hydrogen peroxide

• 1 min : 1mm
• 2 min : 7mm
• 3 min : 10mm
• 4 min : 12mm
• 5 min : 14mm

3 pH - 10ml pH buffer (3), 10 ml grass extract, 10 ml 3% hydrogen peroxide

• 1 min : no froth
• 2 min : no froth
• 3 min : seperation of solutions, no froth
• 4 min : seperations of solutions, no froth
• 5 min : a LOT of seperation

5 pH - 10ml pH buffer (5), 10 ml grass extract, 10 ml 3% hydrogen peroxide

• 1 min : .8mm
• 2 min : 1mm, only at edge
• 3 min : 2mm, thicker at edge
• 4 min : 3.5mm, full layer on top
• 5 min : 5mm

7 pH - 10ml pH buffer (7), 10 ml grass extract, 10 ml 3% hydrogen peroxide

• 1 min : 4mm
• 2 min : 10mm
• 3 min : 15mm
• 4 min : 22mm
• 5 min : 25mm

8 pH - 10ml pH buffer (8), 10 ml grass extract, 10 ml 3% hydrogen peroxide

• 1 min : 15mm
• 2 min : 30mm
• 3 min : 45mm
• 4 min : 55mm
• 5 min : 63mm

9 pH - 10ml pH buffer (9), 10 ml grass extract, 10 ml 3% hydrogen peroxide

• 1 min : 14mm
• 2 min : 35mm
• 3 min : 60mm
• 4 min : 70mm
• 5 min : 80mm

PH 3

PH 5

PH 9

PH 8

Neutral/Control group

PH 7

Conclusions:

Overall we learned a lot from the experiment and it showed us how these pH levels all had different outcomes, rates of reactions, and froth growth when combined with hydrogen peroxide and grass extract. The experiment was fun and exciting, being able to observe the overflows of the 8 and 9 pH solutions’ froth. In conclusion, it was a great experience, and we would love to do the project again.