Lab experiment: The effects of low pH on enzymes
Reactions inside plant and animal cells are catalyzed by a particular class of proteins known as enzymes. Enzymes as catalysts enhance the reaction rate without being altered in any way thus reducing the energy required for reactions to take place. Enzymes normally convert a substrate molecule into a product. This response is called the enzyme-substrate complex and the rate of reaction is influenced by varying a number of factors such as temperature, substrate concentration and the pH. This laboratory experiment is designed to test the effect of an acidic fluid (low pH) on enzymatic activity.
The experiments questions are:
- Does increasing the acidity of an enzymatic reaction increase or decrease the rate of reaction?
- At what pH level is the rate of enzyme (catalase) activity highest and Lowest?
According to the collision theory, chemical reactions are caused by the collision of the reactants particles with enough activation energy to break the pre-existent bonds and the rate of this reaction is increased by increasing the substrate’s concentration among other factors. Therefore, the experiment’s hypothesis is; lowering the pH value of the enzymatic reaction will increase the rate of reaction and hence reducing the reaction time.
The experiment’s substrate is hydrogen peroxide (2H2O2) which is a toxic by-product of cell chemical reactions. The enzyme is catalase a very active cell catalyst found in most aerobic cells, in this experiment a potato tissue. It is responsible for protecting the cells from the toxicity of hydrogen peroxide by rapidly breaking it down into water (H2O) and oxygen (O2).
Hydrogen peroxide + catalase à oxygen + water
The buffer solution’s pH will be varied beginning from a pH of 8.4 and lowering it down to 7.5, 6.5, 5.2 and finally 4.4. The rate of this reaction will be evaluated at every pH level through measuring the rate at which oxygen is produced. This will be achieved by measuring and recording the time taken for the oxygen to reach 10cm3. The other variables such as the quantity of the potato, amount and concentration of hydrogen peroxide and the quantity of the buffer solution will be kept constant for every trial. The experiment is conducted in 3 successive trials and the time for reaction recorded for each pH value. This data is collected in tabular and presented in a graph of pH vs. Reaction time (s).
- Conical Flask
- Hydrogen Peroxide
- Potato (4 grams each)
- pH buffer
- Glass Jar
- Delivery Tube
- Rubber Stopper
Conducting the experiment.
This experiment aims to test and measure the effect of pH on the enzymatic activity of catalase. The pH of the buffer solution used is varied as follows 8.4, 7.5, 6.5, 5.2 and 4.4. The experiment is conducted in 3 trials and for each trial, the time taken for the oxygen produced to reach 10cm3 is recorded in a table. An average reaction time is consequently computed from the three trials for every pH level.
- Set up the apparatus as shown below in the diagram.
- Prepare the buffer solutions in different pH levels to be tested.
- Grate and separate the potato in 5grams portions and place one inside the conical flask.
- Place the delivery tube from the bottom of the conical flask to the measuring cylinder
- Add 20ml of the buffer solution to the conical flask.
- Place the rubber stopper and ensure it is air tight.
- Measure and inject 10ml of 5% hydrogen peroxide solution through the syringe.
- Start the stop clock instantly, and stop once the collected gas reaches 10cm3.
- Repeat the process for each PH value for 3 trials.
- Record the data in a table.
|pH of buffer solution||Trial 1 – time taken to 10cm3 (s)||Trial 2 – time taken to 10cm3 (s)||Trial 3 – time taken to 10cm3 (s)||Average – time taken to 10cm3 (s)|
At the pH of 8.4, the reaction was measured to have an average least reaction time of 7.49 while the pH of 4.4 was measured to have an average least reaction time of 17.28. A reduction in reaction time is observed with the increase of the buffer solution’s acidity.
After conducting the experiment and obtaining the results, it was established that the effect of lowering the pH is the lowering of the catalase activity in the reaction. Therefore it can be concluded that increasing the acidity of an enzymatic reaction decreases the rate of reaction. The effect of pH to speed up the activity of Catalase was found to be stronger at 8.4.
This may be attributed to the phenomenon where lowering the pH consequently weakens the bonds holding the enzyme together thus causing the protein molecules to break apart. This denatures or changes the nature of the enzyme and causes alteration of its polarity within a cell that ultimately deactivates the active sites that recognize the enzyme’s substrate.
Looking back, the experiment’s accuracy could be improved by a set up that can precisely measure the rate of oxygen produced from the reaction per second. For each pH value. Using a digital weighting scale to measure accurately the amount of potato can also improve the experiment’s accuracy.
From the results, my hypothesis was disapproved as pH 4.4 was recorded as the less reactive since more time was taken for the oxygen to reach 10cm3 and pH 8.4 was recorded as the most reactive. Therefore as the acidity increases, the enzyme catalase becomes less reactive.
The results propose that the most conducive pH for catalase to disintegrate hydrogen peroxide into water and oxygen is 8.4 because the least amount of time in seconds was taken to collect the 10cm3oxygen.
Meister, A., Meister, & Alton. (2009). Advances in Enzymology and Related Areas of Molecular Biology. John Wiley & Sons.
Vodopich, D. S., & Moore, R. (2002). Biology laboratory manual. Boston: McGraw-Hill.
Williams, G. (2011). Biology for you. Cheltenham: Nelson Thornes.