enzyme reaction lab

Aakash Parikh

Lab report 2

Mr. Veres

11/23/2011

Factors influencing Enzyme activity

Objective:

To see the changes in enzymatic activity through a series of test and condition , under

which cellular respiration would occur most.

Seeing how different stimuli can affect the outcome of how an enzyme works to catalyze

a reaction; whether it will speed the reaction up immensely or slightly.

Materials:

Water tub

100mL graduate cylinder

Ring stand

Thermometer

Reaction chamber

Yeast ( Catalase)

10mL graduated cylinder

Pipettes

3% hydrogen peroxide

Hot plate

Ice

pH buffer 4,7,10

balance

Distilled Water

NaCl (Salt)

Introduction:

Enzymes are a key part to biological functions in organism. Every organism performs a

series of various chemical reactions every day. These reactions helps the human body function.

Normally, these reactions would process very slow and it would years for the body to break down

glucose because there are catalysts that help slow down the reaction. Biological catalysts are

called enzymes. In this lab we discuss how these different stimuli actually affected the way the

yeast solution respired.

Hypothesis:

Part A) If 10 mL of hydrogen peroxide (H2O2) is combined in a reaction chamber with

1.0 mL of stock catalase solution (yeast) and the reaction rate is measured via the

production of oxygen gas over the course of five minutes, then a control group will be

established by which environmental factors can be analyzed for their influence on

enzymatic activity.

Part B) If 10 mL of hydrogen peroxide (H2O2) is combined in a reaction chamber with

varying concentrations of stock catalase solution (yeast) separately and the reaction rate

is measured via the production of oxygen gas over the course of five minutes, then the

reaction rate will increase as enzyme concentration increases because there are more

active sites for which the substrate molecules can bind. Ultimately, the reaction rate will

level off because the substrate is limiting.

Part C) If 10 mL of hydrogen peroxide (H2O2) is combined in a reaction chamber with 1.0

mL of stock catalase solution (yeast) at varying temperatures and the reaction rate is

measured via the production of oxygen gas over the course of five minutes, then the

reaction rate will increase as the enzyme approaches its optimal temperature and decrease

as the enzyme moves away from its optimal temperature.

Part D) If 10 mL of hydrogen peroxide (H2O2) at three pH values is separately combined

in a reaction chamber with 1.0 mL of stock catalase solution (yeast) and the reaction rate

is measured via the production of oxygen gas over the course of five minutes, then the

reaction rate will increase as the enzyme approaches its optimal pH and decrease as the

enzyme deviates from its optimal pH.

Part E) If 10 mL of hydrogen peroxide (H2O2) at varying concentrations is separately

combined in a reaction chamber with 1.0 mL of stock catalase solution (yeast) and the

reaction rate is measured via the production of oxygen gas over the course of five

minutes, then the reaction rate will increase as substrate concentration increases because

there will be more opportunities for the substrate molecules to bind to the active sites.

Ultimately, the reaction rate will level off because all of the active sites will be filled.

Part F) If 5 mL of hydrogen peroxide (H2O2) is mixed with 5 mL of varying

concentrations of NaCl independently, combined in a reaction chamber with 1.0 mL of

stock catalase solution (yeast), and measured for the reaction rate via the production of

oxygen gas over the course of five minutes, then the reaction rate will increase as the

enzyme approaches its optimal salinity and decrease as the enzyme diverges from its

optimal salinity.

II. Procedure:

Part A) The Time Course of Enzyme Activity

1. Fill a plastic rectangular container ¾ full of tap water. Submerge the graduated

cylinder to fill most of it with water. Turn the graduated cylinder upside down to keep

the water in. Suspend it in the clamp on the ring stand. Adjust the height of the clamp

on the ring stand so the open end of the graduated cylinder is about 3 cm above the

bottom of the container. Record the temperature of the water. Refer to the labeled

diagram to the right for concerns or

questions, but do not submerge the reaction chamber yet.

2. Obtain a reaction chamber, 10 mL of 3% hydrogen peroxide (H2O2) solution, and

some stock catalase solution (yeast).

3. Pour the 10 mL of hydrogen peroxide (H2O2) into the reaction chamber. Pipette in 1.0

mL of stock catalase solution (yeast). Stopper the reaction chamber tightly, submerge

it in the water bath, and place the plastic tubing into the bottom of the graduated

cylinder so that the bubbles formed in the reaction chamber are captured by the

inverted graduated cylinder.

4. Measure the gas level in the graduated cylinder initially and at 30-second intervals for

five minutes. The data should be plotted on a graph.

Part B) The Effect of Enzyme Concentration on Enzyme Activity

1. Repeat Part A, but this time use three different levels of enzyme concentration as

follows:

- 75% concentration: Pipette in 0.75 mL of catalase solution in the reaction chamber

instead of 1.0 mL.


instead of 1.0 mL.


instead of 1.0 mL.

2. Data recorded from the varying enzyme concentrations should be plotted on a graph.

Part C) The Effect of Temperature on Enzyme Activity

1. Repeat Part A, but this time use three different temperatures as follows:

- 5°C: Set up your water bath and add ice so that it is chilled to 5°C for five minutes

before running the experiment. Keep adding ice to maintain the cool temperature.

- 37°C: Set up your water bath with heated water so that it is warmed to 37°C for five

minutes before running the experiment. Keep adding hot water to maintain the warm

temperature.

- 100°C: Rather than using a water bath of boiling water, boil the catalase solution for

five minutes. Let the catalase cool and then run the experiment in room temperature

water.

2. Data recorded from the varying temperatures should be plotted on a graph.

Part D) The Effect of pH on Enzyme Activity

1. Repeat Part A, but this time use 1.5% hydrogen peroxide solutions at three different

pH values as follows:

- pH 4: Add 5 mL of H2O2 to 5 mL of pH 4 buffer.



2. Data recorded from the solutions at varying pH values should be plotted on a graph.

Part E) The Effect of Substrate Concentration on Enzyme Activity

1. Repeat Part A, but this time use four different substrate concentrations as follows:

- 0%: Use 10 mL of distilled water only.

- 0.3%: Prepare this by adding 3 mL of H2O2 to 7 mL of distilled water.

- 1.5%: Prepare this by adding 5 mL of H2O2 to 5 mL of distilled water.

- 3.0%: Use the data from Part A.

2. Data recorded from the varying substrate concentrations should be plotted on a graph.

Part F) The Effect of Ionic Concentration on Enzyme Activity

1. Repeat Part A, but this time mix 5 mL of hydrogen peroxide solution with three

different ionic concentrations as follows:

- 10% NaCl: Dissolve 5 g of NaCl in 50 mL of water and add this solution to 5 mL of

H2O2.

- 2% NaCl: Dissolve 1 g of NaCl in 50 mL of water and add this solution to 5 mL of

H2O2.

- 0% NaCl: Add 5 mL of distilled water to 5 mL of H2O2.

Data Part A and B1 mL of yeast:30 sec: 4mL1 min: 6mL1.30: 9mL2 min: 12mL2.30: 15.5 mL3 min: 18 mL3.30: 21 mL4 min: 22 mL4.30: 24.5 mL5 min: 26 mL.75 ml of yeast:30 sec: 1mL1 min: 4mL1.30: 5.5mL2 min: 7mL2.30: 11 mL3 min: 16 mL3.30: 20 mL4 min: 22 mL4.30: 23.5 mL5 min: 24.5 mL5 ml of yeast:30 sec: 1mL1 min: 3mL1.30: 4mL2 min: 4.5 mL2.30: 4.7 mL3 min: 5 mL3.30: 5.5 mL4 min: 5.7 mL4.30: 6 mL5 min: 6.3 mL.25 mL of yeast30 sec: .7 mL1 min: 1 mL1.30: 1.3 mL2 min: 1.5 mL2.30: 1.8 mL3 min: 2 mL3.30: 2.2 mL4 min: 2.4 mL4.30: 2.6 mL5 min: 2.7 mL

Part C

5* C

30 sec: .1mL

1 min: .1 mL

1.30: .2 mL

2 min: .2 mL

2.30: .2 mL

3 min: .3 mL

3.30: .3 mL

4 min: .4 mL

4.30: .4 mL

5 min: .5 mL

37*C

30 sec: 2 mL

1 min: 5 mL

1.30: 7.3 mL

2 min: 8.4 mL

2.30: 13 mL

3 min: 15 mL

3.30: 17 mL

4 min: 19.4 mL

4.30: 20.2 mL

5 min: 22.4 mL

100*C

30 sec: 1 mL

1 min: 1.5 mL

1.30: 1.8 mL

2 min: 1.9 mL

2.30: 2.0 mL

3 min: 2.1 mL

3.30: 2.2 mL

4 min: 2.3 mL

4.30: 2.4 mL

5 min: 2.5 mL

Part D

PH 4

30 sec: 2 mL

1 min: 3.5 mL

1.30: 4.1 mL

2 min: 4.7 mL

2.30: 5.1 mL

3 min: 5.3 mL

3.30: 5.6 mL

4 min: 5.8 mL

4.30: 6 mL

5 min: 6.3 mL

PH 7

30 sec: 2 mL

1 min: 3.9 mL

1.30: 5 mL

2 min: 5.9 mL

2.30: 6.1 mL

3 min: 6.8 mL

3.30: 7.3 mL

4 min: 7.9 mL

4.30: 8.4 mL

5 min: 9 mL

PH 10

30 sec: 1 mL

1 min: 1.5 mL

1.30: 1.9 mL

2 min: 2.1 mL

2.30: 2.3 mL

3 min: 2.7 mL

3.30: 2.9 mL

4 min: 3 mL

4.30: 3.2 mL

5 min: 3.3 mL

Part E

0% substrate30 sec: 2 mL1 min: 3 mL1.30: 3.7 mL2 min: 4 mL2.30: 4.2 mL3 min: 4.3 mL3.30: 4.3 mL4 min: 4.4 mL4.30: 4.5 mL5 min: 4.5 mL

.3% substrate30 sec: 2.3 mL1 min: 4.7 mL1.30: 7 mL2 min: 9 mL2.30: 11 mL3 min: 13 mL3.30: 15 mL4 min: 15.7 mL4.30: 17 mL5 min: 19 mL

1.5% substrate30 sec: 2.5 mL1 min: 6 mL1.30: 9 mL2 min: 12 mL2.30: 14.3 mL3 min: 6.2 mL3.30: 18.1 mL4 min: 19.3 mL4.30: 21 mL5 min: 23.4 mL

3% substrate30 sec: 4mL1 min: 6mL1.30: 9mL2 min: 12mL2.30: 15.5 mL3 min: 18 mL3.30: 21 mL4 min: 22 mL4.30: 24.5 mL5 min: 26 mL

Part F

10% NaCl

30 sec: 1mL

1 min: 1.5mL

1.30: 2mL

2 min: 2.1mL

2.30: 2.2 mL

3 min: 2.3 mL

3.30: 2.4 mL

4 min: 2.8 mL

4.30: 3.0 mL

5 min: 4 mL

2% NaCl

30 sec: 5mL

1 min: 8.5mL

1.30: 13.5mL

2 min: 17mL

2.30: 20 mL

3 min: 23 mL

3.30: 26 mL

4 min: 29 mL

4.30: 31 mL

5 min: 35 mL

0% NaCl

30 sec: 2.5 mL

1 min: 6 mL

1.30: 9 mL

2 min: 12 mL

2.30: 14.3 mL

3 min: 6.2 mL

3.30: 18.1 mL

4 min: 19.3 mL

4.30: 21 mL

5 min: 23.4 mL

Conclusion:

Part A)

The increase in the gas level or the amount of O2 evolved was 8 mL over the course of

five minutes.

The reaction rate, which is the change in gas level divided by the change in time for

this part of the laboratory and all future parts, is the control group for enzyme

concentration (100%), temperature (24°C), and substrate concentration (3.0%).

This part of the laboratory allows us to introduce environmental conditions, or

independent variables, in future parts and conclude how they affect the rate of enzyme

activity.

Part B)

For 100% enzyme concentration, the increase in the gas level or the amount of O2

evolved was 8 mL over the course of five minutes.







The enzyme concentration is directly proportional to the rate of enzyme activity. In

layman’s terms, as the enzyme concentration increases, the rate of the enzyme activity

does as well.

Part C)

For the reaction at 24°C, the increase in the gas level or the amount of O2 evolved was

8 mL over the course of five minutes.

For the reaction at 5°C, the increase in the gas level or the amount of O2 evolved was 2

mL over the course of five minutes.

For the reaction at 37°C, the increase in the gas level or the amount of O2 evolved was

20 mL over the course of five minutes.

For the reaction at 100°C, the increase in the gas level or the amount of O2 evolved

was 2 mL over the course of five minutes.

There is no direct correlation between temperature and the rate of enzyme activity.

Ergo, the stock catalase solution (yeast) has an optimal temperature at which it

operates best. Based on the data, the optimal temperature must be somewhere in the

range of 37°C.

Part D)

For the reaction at the pH of 4, the increase in the gas level or the amount of O2






The pH value is inversely proportional to the rate of enzyme activity. In layman’s

terms, as the pH value increases (more basic), the rate of enzyme activity decreases.

Keep in mind, however, that the full range of pH values was not tested. Therefore,

there may be no correlation between pH value and the rate of enzyme activity. Instead,

there could be an optimal pH value somewhere in the range of pH 4.

Part E)

For 3.0% substrate concentration, the increase in the gas level or the amount of O2


For 0% substrate concentration, the increase in the gas level or the amount of O2

evolved was 0 mL over the course of five minutes. This was because there was no

substrate in the form of hydrogen peroxide to decompose into oxygen gas.





The substrate concentration is directly proportional to the rate of enzyme activity. In

layman’s terms, as the substrate concentration increases, the rate of enzyme activity

does as well.

However, the reaction rate for 1.5% substrate concentration and 3.0% substrate

concentration are equal because the total number of enzymes and active sites becomes

a limiting factor.

Part F)

For 10% NaCl concentration, the increase in the gas level or the amount of O2 evolved






The ionic concentration, or salinity, is inversely proportional to the rate of enzyme

activity. In layman’s terms, as the salinity increases, the rate of the enzyme activity

decreases.

Keep in mind, however, that the full range of salinities was not tested. Therefore, there

may be no correlation between salinity and the rate of enzyme activity. Moreover, the

reaction rate of 2% and 0% are very close to one another. There could be an optimal

salinity somewhere in the range of 0-2%.