Patsornkarn Vorapharuek

October 25, 2009

Biology SL

Effect of pH on Enzyme Activity

Introduction:

Enzymes require specific conditions to work effectively. An enzyme will have an optimum pH where enzyme activities occur at the fastest rate. In this investigation, the optimum pH will be investigated for chicken liver enzymes. By placing various hydrogen peroxide solutions of different pH in catalase which is the enzymes, the rate of reaction can be collected and therefore aids the determination of the optimum pH level. The solutions contain a pH level of 3, 7, 9 and 11. The enzyme combined with pH solution creates a reaction. The rate of this reaction can be recorded with a pressure sensor.

It is essential to understand the functions of enzymes during this investigation. Enzymes are biological catalysts made in living organisms. (Biology, Allot) Enzymes are proteins that catalysts chemical reactions. They are generally specific, catalyzing few reactions. In chemical reactions, substrates are the reactant. Since enzymes are specific there are only few possible substrates, this is known as the enzyme-substrate specificity. The substrate binds to the active site where only specific molecules can fit. The way an enzyme and substrate fits together is like a lock and key model. In this lab, the enzyme is the chicken liver while the substrate is the H2O2. Enzymes are easily capable of being damaged due to various conditions; this is called denaturing. When enzymes are denatured, the structure of the protein changes permanently, therefore no longer carrying out its functions. Factors such as temperature, pH and substrate concentration affects enzyme activities. The optimum pH for the majority of enzymes is the pH of 7.

By placing the substrate of h2o2 with catalase, a chemical reaction occurs. The hydrogen peroxide is catalyzed by the enzymes, creating oxygen and water in the reaction. This can be shown by the following equation.

H2O2 -------> 2 H2O + O2

In the investigation, a pressure sensor is used. This pressure sensor detects the release of oxygen in the test tube. Therefore, being measured in kPa per second.

pH level, the independent variable of this investigation is a measure of the degree of acidity or alkalinity of a solution. It is measured on a scale of 0-14. The pH of 7.0 represents neutrality. Substances with pH level below 7 are acidic while substances above seven are alkaline. (Parris-supply.com)

The following is the expected graph of enzyme activity (figure 1). As pH increases or decreases from the optimum pH; the rate of enzyme activity is lower.

What is the optimum pH level for chicken liver enzymes?

Figure 1: The expected graph of enzyme activity rate due to changes of pH level. (Worthington-biochem.com)

Independent and Dependent Variable:

The independent variable will be the pH level of the h2o2 solution. In this investigation, buffers of pH level 3,7, 9 and 11 are placed in h2o2 creating h2o2 of those pH levels. These various solutions of h2o2 mixed with catalase will create a chemical reaction. The dependent variable is the rate of these reactions.

Controlled Variables:

Factor Importance Method of control

Amount of enzyme The amount of enzymes determines the amount of chemical reaction possible to occur

Keeping a constant amount of enzymes in each trial (10 drops)

Temperature Temperature is one of the key factors that affect enzyme activity; therefore capable of effecting results of this lab if different for each trial

The investigation is conducted in one location which has a constant temperature

Ratio of buffer to H2O2 When an equal amount of buffer is added to H2O2or other solutions, the pH level of the solutions changes to that of the buffer. If the buffer and H2O2ratio are unequal then the pH of the solution may not be as needed

To keep these measurements constant and equal, 5ml of H2O2and buffer are always measured with a graduated cylinder

Same enzymes throughout the investigation

After a period of time, enzymes break down therefore not having the same properties. This would affect the chemical reactions that would occur.

To control this, the investigation is done all in a short period of time with the same set of enzymes.

Test tube size A pressure sensor functions by measuring the pressure of the gas produced by the chemical reaction. If a test tube is bigger or smaller, the air pressure would not be constant.

Test tubes of the same size were used to conduct all trials.

Procedure:

It is essential that all materials are prepared before the investigation. Materials include the following

Beakers

Graduated cylinders

Disposable pipettes

Test tubes

Chicken liver (blended)

H2O2

Buffer (pH of 3,7,9,11)

Pressure sensor connected to logger pro

Once all the materials are prepared, place 10 drops of enzymes in 16 test tubes. Following that, measure 5 ml of H2O2for 16 other test tubes. Since there are four different buffers, mix 5 ml of each buffer in four test tubes of H2O2. This will result in four test tubes of H2O2with a level 3 pH, four with level 7 pH, four with level 9 pH and four with level 11 pH. Once everything is measured, prepare

the pressure sensor.

To begin the collection of chemical reaction rates, press start on Logger Pro to activate the pressure sensor. Next, pour the H2O2 into the liver and tightly plug the pressure sensor onto the test tube. Stop the collection once the enzyme has denatured. Repeat these steps with all the trials and record the rate. The rate can be found on the Logger Pro graph. By using linear fit on the slope, the pressure in kPA will be shown.

Figure 2: H2o2 solution

Figure 3: Chicken liver enzymes (catalase) placed in test tubes

Figure 4: Shows H2O2solution with pH of 3

Data Collection and Processing:

Table 1: The Rate of Chemical Reaction in Various pH

Data Collection Data Processing

Trial 1 (kPa) Trial 2 (kPa) Trial 3 (kPa) Trial 4 (kPa) Uncertainty Average (kPa)

pH 3 0.41 0.30 0.23 0.20 ±0.11 0.29

pH 7 1.87 2.35 1.98 2.13 ±0.24 2.08

pH 9 5.30 5.21 6.07 5.50 ±0.43 5.52

pH 11 4.05 4.13 4.26 6.25 ±1.10 4.67

This table demonstrates the raw data collected. It is demonstrated according to each pH to each trial. Notice that the measurements of the rates are in Kilo Pascals (kPa) per second. Furthermore, the uncertainties of each pH are shown. Also, the data is processed and the averages are found for each pH.

Sample Calculations:

Uncertainties

The uncertainty of the pressure in kPa is the range of the data, divided by two.

Example on pH 3: [(0.41-0.20) / (2)] = Uncertainty

(.21)/(2) = 0.105 kPa

Therefore, the uncertainty for data collected for the trial of pH 3 is ±0.11 kPa

Average per pH

Adding all the data per pH and dividing by the amount of data can find the average of each set of pH.

Example on pH 3: [(0.41 + 0.30 + 0.23 + 0.20) / (4)]

(1.14) / (4) = 0.29 kPa

Therefore, the average data of pH 3 is 0.29 kPa

Table 2: Qualitative Observations

Observations

pH buffers The solutions each had a different color but all were transparent

Blended chicken liver enzymes (catalase)

Orange color. Opaque

Mixture of H2o2 and enzyme

Bubbles formed at the top. Murky color

This table shows the physical appearances of the materials involved in this investigation.

Figure 5: Demonstrates the average rate of reaction in kPa per second for each pH level. Notice that the pH of 3 has the lowest rate of reaction. Following that, the rate of reaction increases as pH increases but the rate begins to fall after pH 9. Error bars are incorporated to show possible rates due to inefficiency of lab conduction.

Conclusion:

Through this investigation, the optimum level of pH for catalase was found. From the collected data, the rate of reaction was quickest at pH level 9. The rate of reaction increased as pH increased; reaching its peak at pH 9 and decreasing at pH 11. This can be observed in figure 5, notice the inclining slope before pH 9 then the declining slope following that. The expected graph shown in figure 1 also demonstrated an increase in rate and then a decrease after the optimum level is reached. Uncertainties were calculated for the data collected. From the t-test done on Graphpad.com, all data sets were significantly different except for pH 9 and pH 11. This maybe due to the data in pH 11, the third fourth trial produced a result that was unexpected and far off the other three trials; possibly due to error in conduction.

Evaluation:

Many sources of error could be improved in the future to further guarantee accuracy. First, the catalase was measured with a disposable pipette. Ten drops of catalase were placed in each test tube; the source of error is that the disposable pipette relies on the investigator to create the drops. Therefore, it is very easy for the catalase to be distributed unevenly. In order to fix this, catalase should be measured to the ml, similar to the measuring of the solutions.

In addition, since materials were shared between multiple classes, a lot of the test tubes were partially wet. Often, during the investigation, drying them was a priority but when time was short; some were left a bit wet. This could have deluded the catalase. In order to prevent this in the future, in preparation for the lab; the test tubes should be carefully dried before placing any solution or enzymes in them.

Another source of error was the rubber stopper of the pressure sensor. How far the stopper is pushed in the test tube affects the results. This was an issue because sometimes the stopper would not be pushed in all the way, giving in oxygen from outside the test tube, which would have an effect on the pressure inside the test tube. This is a limitation because the sizes or the stoppers did not fit the test tubes well. There were different sizes available but since there were many groups, choice was limited. In order to prevent this, the size should be fitted and tried carefully before.

Lastly, the angle that the test tubes were placed at could affect the results. Many times, the test tubes were held by hand. For example, when placing catalase in the test tube, the angle could have caused the catalase to run down the side; therefore changing the amount of enzymes that would be mixed with the substrate. This could be fixed by keeping the test tubes in a test tube rack at all times; which would maintain the test tubes in vertical position.