Scientific Inquiry

Activity: Give a brief outline of what you are planning to do. This experiment investigates the effect of 5 different pH conditions on enzyme activity on breaking down hydrogen peroxide and observes the catalytic action of 2 sources of catalases on hydrogen peroxide. Are there possible risks? Consider the following:


Background information/Introduction
What are enzymes?
Enzymes are biological catalysts that speed up biochemical reactions by lowering their activation energy (Figure 1).They play an important role in cellular metabolism.They either split a substrate apart or bind a substrate together.Most enzymes are wholly or partially made of proteins.Types of enzymes include catalase, diastase, pectinase and so on.Enzymes can be used repeatedly because they are not consumed during the reaction.Enzymes increase the rate of reaction but have no effect on the concentrations of reactants and products at equilibrium.

Figure 1. Differences in activation energy between reactions with and without enzyme
Reference: https://www2.nau.edu/lrm22/lessons/enzymes/enzymes.htmlAdditionally, enzymes are very specific for their substrates in vivo, for example, hydrogen peroxide only reacts with catalase.The active site of an enzyme is the location on the enzyme where substrate binds.Induced fit model explains that this complementary binding where both enzyme and substrate change shape a little bit so that they bind together at its maximum strength at the transition state of a catalytic reaction (Table 1).

Stages of enzyme catalysis Stage 1:
First thing that happen in an enzyme-substrate reaction.
The substrate is yet to come into contact.

Stage 2:
The enzyme binds with the substrate, but this binding is not perfect.The forces holding the enzyme and the substrate is strong, but they are not in their maximum strength yet.They do not fit like puzzle pieces.

Stage 3:
Both enzyme and the substrate change shape a little bit and binds together at its maximum strength.The enzyme is catalysing at its full force.

Stage 4:
The enzyme is separating the substrate into two parts.

Stage 5:
Products of the reaction are released from the enzyme and the enzyme is returned to its initial state as in stage 1.

Factors affecting enzyme activity
The factors that affect the enzyme activity on a given substrate are potential hydrogen (pH), temperature, enzyme and substrate concentrations and presence of enzyme-specific inhibitors or activators.Enzymes are highly sensitive and function optimally at their favourable pH, temperature, enzyme and substrate concentrations, and in the absence of enzyme-specific inhibitors.PH is a scale measure whether a chemical is acidic or basic and describes how much ionic hydrogen (H+) or hydroxide (OH) it contains.In this experiment, pH will be investigated on the rate of enzyme-catalysed reaction.

What is hydrogen peroxide?
The substrate used in this experiment is hydrogen peroxide which is a pale blue liquid with a chemical formula of H2O2.It has antiseptic properties and can be used as an oxidising and bleaching agent.It is also a poisonous by-product of cellular metabolism produced by living cells.The enzyme catalase splits hydrogen peroxide apart to produce oxygen and water.

Aim
This experiment investigates the effect of pH on catalase activity and observes the catalytic action of two sources of catalase on hydrogen peroxide.

Hypotheses
Hypothesis 1: If the pH is below or above the optimum pH, the rate of the catalytic action of catalase will decrease.
Hypothesis 2: Animal cells contain more catalase than plant cells.Chicken liver that contains higher catalase concentration per unit volume has a faster rate of catalytical reaction than potato.

Independent Variables
The pH of the hydrogen peroxide solutions and the source of catalases are the independent variable of this experiment.

Dependant Variable
The amount of gas pressure exerted by oxygen produced is the dependant variable of this experiment.The rates of reaction of decomposition of hydrogen peroxide can be compared by measuring the gas pressure using PASCO PASPORT Chemistry Sensor.The higher the gas pressure exerted by oxygen produced at a given time interval, the faster the catalytic reaction is.

Controlled Variables
Controlled variables are to ensure that this experiment is a 'fair test' (Table 2).

Controlled variables Method of Control Reason Number and volume of potato and chicken liver
The potato and chicken liver cubes are measured with a plastic ruler and cut to a volume of 1 cm 3 using a sharp knife. 1 potato or chicken liver cube is placed in the conical flask of each pH solution.
The potato and chicken liver are cut to the same volume to compare and determine which source of catalase has higher concentration per unit volume.Different number of potato or chicken liver cubes can affect the rate of reaction.Concentration and volume of hydrogen peroxide in each pH solution Each pH solution contains the same amount of substrate, i.e., 30 ml of 6% hydrogen peroxide.
Different concentration and volume of hydrogen peroxide will affect the rate of the catalase activity.The amount of substrate must be the same across experiments for comparison.

The same potato is used
The potato cubes used in this experiment are cut from the same potato.

Different potatoes may have different concentration of catalase. Measuring time for gas pressure
The data is collected at the specific times, i.e., 30 sec, 60 sec, 90 sec and 120 sec.
The data collected must be measured at the same time for comparison.PH conditions of the substrate The pH conditions of the substrate for both chicken liver and potato cubes are the same, i.e., pH 1.6, 3.3, 6.5, 9.9 and 11.4.
The same pH conditions are compared across both chicken liver and potato catalases.

Size of conical flask
The 100 ml conical flasks of the same size are used.
Conical flasks must be of the same size to ensure the same height of the solution for fair comparison.The opening of the conical flasks must be of the size to ensure good fitting of the stopper to prevent leaking of the oxygen gas.PASCO PASPORT Chemistry MultiMeasure Sensor and stopwatch.
The same equipment is used for measurement.This is to minimise systematic errors and hence improve the accuracy of the data collected.

Room Temperature
All the experiments are conducted at the room temperature of 22 °C.
Different temperature affects the enzyme activity.

Uncontrolled Variables
After the catalase is placed into the conical flask, the stopper of the gas pressure tubing must be connected to the conical flask immediately and the stopwatch must be activated simultaneously.The time lapse between placing the catalase into the conical flask and connecting the stopper of the gas pressure tubing can be an uncontrolled variable.However, it is minimised by shortening the time of the action.The time difference is within 1 second.There are no other significant uncontrolled variables in this experiment.Put on personal protective equipment: apron, safety glasses, safety gloves, enclosed footwear, surgical mask.2. Prepare all the equipment and materials.3. Set up the chemistry sensor with the pH probe and the gas pressure tubing.4. Cut 5 1cm 3 cubes of potato and chicken liver with a ruler and a sharp knife.

Equipment and Materials
Step 2: Prepare 100 ml of pH hydrogen peroxide solutions.pH 1.6 1. Rinse the 100 ml measuring cylinder with hydrogen peroxide to prevent dilution.2. Fill the measuring cylinder with 60 ml 6 % hydrogen peroxide.3. Fill the measuring cylinder with 30 ml of distilled water.4. Using the pH probe, fill the measuring cylinder with 1 mole HCL until pH is 1.6. 5. Fill the measuring cylinder with distilled water until the total volume of the solution is 100 ml. 6. Use the stirring rod to slowly mix the solution evenly.7. Label the solution of pH 1.6 with an adhesive labelling sticker.pH 3.3 1. Rinse the 100 ml measuring cylinder with hydrogen peroxide to prevent dilution.2. Fill the measuring cylinder with 60 ml 6 % hydrogen peroxide.3. Fill the measuring cylinder with 38 ml of distilled water.4. Test the pH using the pH probe which is about pH 5.

Fill the measuring cylinder with drops of 1 mole hydrochloric acid
until the pH reaches pH 3.3.6. Fill the measuring cylinder with distilled water until the total volume of the solution is 100 ml. 7. Use the stirring rod to slowly mix the solution evenly.8. Label the solution of pH 3.3 with an adhesive labelling sticker.pH 6.5 1. Rinse the 100 ml measuring cylinder with hydrogen peroxide to prevent dilution.2. Fill the measuring cylinder with 60 ml 6 % hydrogen peroxide.3. Fill the measuring cylinder with 30 ml of distilled water.4. Using the pH probe, fill the measuring cylinder with 1 mole sodium hydroxide until pH is 6.5. 5. Fill the measuring cylinder with distilled water until the total volume of the solution is 100 ml. 6. Use the stirring rod to slowly mix the solution evenly.7. Label the solution of pH 6.5 with an adhesive labelling sticker.pH 9.9 1. Rinse the 100 ml measuring cylinder with hydrogen peroxide to prevent dilution.
3. Fill the measuring cylinder with 30 ml of distilled water.4. Using the pH probe, fill the measuring cylinder with 1 mole sodium hydroxide until pH is 9.9. 5. Fill the measuring cylinder with distilled water until the total volume of the solution is 100 ml. 6. Use the stirring rod to slowly mix the solution evenly.7. Label the solution with pH 9.9 with an adhesive labelling sticker.pH 11. 4 1.Rinse the 100 ml measuring cylinder with hydrogen peroxide to prevent dilution.2. Fill the measuring cylinder with 60 ml 6 % hydrogen peroxide.3. Fill the measuring cylinder with 35 ml of 1 mole sodium hydroxide.4. Using the pH probe, test if the pH is 11.4. 5.If the pH is not 11.4,then add drops of 2 mole sodium hydroxide until the pH is 11.4.6. Fill the measuring cylinder with distilled water until the total volume of the solution is 100 ml. 7. Use the stirring rod to slowly mix the solution evenly.8. Label the solution of pH 11.4 with an adhesive labelling sticker.
Note: Make sure the pH probe is rinsed with distilled water before testing each solution.

Potato catalase
1. Rinse each conical flask with each pH solution to prevent dilution.2. Fill each 100 ml conical flask with 50 ml of each pH solution.3. Label each pH solution of pH 1.6, pH 3.3, pH 6.5, pH 9.9 and pH 11.4 with adhesive labelling stickers.4. Place a 1cm 3 potato cube in the conical flask for pH 1.6 solution.5. Immediately place the stopper of the gas pressure tubing in the conical flask to measure the increase of gas pressure at time intervals of 30 sec, 60 sec, 90 sec and 120 sec.6. Record data between time intervals.7. Repeat steps 4 to 6 for the other four pH solutions of pH 3.3, pH 6.5, pH 9.9 and pH 11.4.

Chicken liver catalase
1. Rinse each conical flask with each pH solution to prevent dilution.2. Fill each 100 ml conical flask with 50 ml of each pH solution.
3. Label each pH solution of pH 1.6, pH 3.3, pH 6.5, pH 9.9 and pH 11.4 with adhesive labelling stickers.4. Place a 1cm 3 chicken liver cube in the conical flask for pH 1.6 solution.5. Immediately place the stopper of the gas pressure tubing in the conical flask to measure the increase of gas pressure at time intervals of 30 sec, 60 sec, 90 sec and 120 sec.6. Record data between time intervals.7. Repeat steps 4 to 6 for the other four pH solutions of pH 3.3, pH 6.5, pH 9.9 and pH 11.4.

Risk Assessment Safety Precautions
Prudent laboratory safety practices were followed.Chemical contacts were avoided by putting on personal protective equipment including an apron, safety glasses, safety gloves, enclosed footwear and a surgical mask for preventing inhalation of chemicals.Hair is tied back so that hair does not contact with any chemicals.The experiment was handled with care as the chemicals (H2O2, HCL, NaOH) are extremely corrosive.The chemicals can cause serious eye damage, shortness of breath if inhaled and skin irritations as well as burns on skin.When pouring the pH solutions, a slow pouring action was used to minimise the risk of spilling of chemicals.When cutting the potato and chicken liver into cubes, the knife was carefully handled to prevent cuts.Experiments were done in a well-ventilated laboratory.
The equipment and apparatus used in this experiment were carefully handled to prevent any accidents.

Environmental Consideration
The experiment was conducted in compliance with the control measures for preparation, usage of laboratory materials and disposal of chemical wastes.There were no significant environmental considerations as the equipment and actions used in this experiment presented no hazard or danger to the environment.

Ethical Consideration
There were no significant ethical considerations as the equipment and actions used in this experiment presented no harm to society or any individual.

Processing and Analysing Data and Information:
Table 3. Experiment 1: The rate of reaction of potato catalase decomposing hydrogen peroxide.Chicken liver catalase pH vs gas pressure in kPa at 120 sec Chicken liver catalase is 12.9 times faster in the rate of reaction than potato catalase at their optimum pH.
The rates of reaction for the potato and chicken liver catalases decomposing hydrogen peroxide in five different pH conditions (pH 1.6, 3.3, 6.5, 9.9 and 11.4) are compared.At 120 seconds, the potato catalase shows the greatest increase of gas pressure of 4.6 kPa at pH 9.9, followed by pH 11.4, pH 6.5, pH 3.3.The least increase of gas pressure for potato catalase is 1.5 kPa at pH 1.6.Based on the graph, the optimum pH for potato catalase is pH 9.9 at which it produces the greatest rise of gas pressure of 4.6 kPa at 120 seconds (Table 3, Table 5, Figure 2b).
Comparatively, at 120 seconds, the chicken liver catalase shows the maximum increase of gas pressure of 59.3 kPa at pH 6.5, followed by pH 3.3, pH 9.9, pH 1.6.Chicken liver catalase shows the minimum increase of 7.0 kPa at pH 11.4.Based on the graph, Chicken liver catalase works best at the optimum pH of 6.5 at which it produces the greatest rise of gas pressure of 59.3 kPa at 120 seconds (Table 4, Table 6, Figure 3b).
In the interval of 2 minutes, both catalases show constantly increased rate of reaction.Both catalases exhibit similar trend in change of gas pressure kPa at time intervals of 30, 60, 90 and 120 sec respectively (Figure 2a, Figure 3a).
Interestingly, there is a huge difference in rate of reaction between potato catalase and chicken liver catalase.Chicken liver catalase decomposes hydrogen peroxide more efficiently than potato catalase as more oxygen is produced during the enzyme-catalysed reactions (59.3 vs 4.6 kPa at 120 seconds) (Table 7, Table 8).In other words, chicken liver catalase is much more powerful than potato catalase as chicken liver catalase works 12.9 times faster in the rate of reaction than potato catalase at their optimum pH.
Additionally, the maximum increase of gas pressure in potato catalase at its optimum pH is lower than the minimum increase of gas pressure in chicken liver catalase at its least favourable pH (4.6 vs 7.0 kPa at 120 seconds) (Table 7, Figure 4).The higher the concentration of an enzyme is, the faster the catalytic reaction is.This finding implies that chicken liver contains much higher concentration of catalase per unit volume than potato.

Data discussion
The result of the investigation supports the hypothesis that the rate of enzyme activity will reduce when the pH is below or above the optimum pH.All enzymes have an optimum pH at which they function best.Enzymes also have a working range of pH values at which they can still function well.The active site is formed by a specific conformation of the enzyme's amino acid side chains, which has a specific ionization state that forms the resulting specific three-dimensional structure of the enzyme.Changes in pH affects the ionization state of the amino acids which affects the ionic bonds that hold the three-dimensional shape of the enzyme, thereby impacting the effectiveness of the enzyme activity.In living systems, biochemical buffers maintain an optimum pH range for the enzyme to function best.Extreme pH conditions will cause enzyme to denature.Denaturation is a permanent and irreversible change caused by the breaking of the hydrogen and ionic bonds that maintain the three-dimensional shape of the enzyme.Similar to high temperature, acidic and basic pH can disrupt the three-dimensional shape of the enzyme and change the shape of the active site and therefore causing an irreversible change in the function of the enzyme.When the enzyme becomes denatured and loses its structure, the substrate will not bind to the denatured active site (Figure 5).On the other hand, pH also affects the charge and the shape of the substrate, therefore the substrate cannot bind to the active site.The result also supports the hypothesis that animal cells have more catalase than plant cells.In this experiment, chicken liver has higher concentration of catalase per unit volume than potato.Catalase is found in all aerobic organisms including animal cells, plant cells and human cells.Animal cells have a higher rate of cellular respiration than plant cells, therefore requiring more catalase to decompose hydrogen peroxide which is a toxic metabolic by-product of the cellular respiration.

Extension of knowledge
Enzymes can accelerate the rate of catalytic reaction by 10 6 to 10 15 folds.Catalase is an antioxidant enzyme which can be found in abundance in liver where toxin is removed.According to Price and Greenfield (1954), rat liver has a range of 160 to 180 units of catalase per gram.In plants, catalase is involved with photorespiration and photosynthesis in peroxisome organelles where hydrogen peroxide is decomposed into water and oxygen gas.There are three types of catalases which are monofunctional heme-containing catalases, heme-containing catalase-peroxidases, and manganesecontaining catalases (Figure 5).One molecule of catalase can decompose millions of hydrogen peroxide molecules per minute.Extremophiles have special enzymes called extremozymes that help them to survive.Those enzymes have other applications to our society.For example, thermophiles possess amylases which have many industrial uses such as baking and making paper.
Acatalasemia, a genetic disorder associated with catalase deficiency, is caused by mutations in CAT gene that gives instructions for producing catalase.Study estimates that 1 in 31,250 of individuals in general population have acatalasemia.Low level of catalase activity results in insufficient decomposition and excessive accumulation of hydrogen peroxide in the body.Acatalasemia not only cause body cells to be susceptible to oxidative damage to DNA, proteins and lipids resulting in oxidative stress, but also increases a higher risk of disease and health problems including diabetes mellitus and atherosclerosis.Moreover, a build-up of hydrogen peroxide due to a lack of catalase may contribute to the development of grey hair.Other diseases related to enzyme deficiency include Phenylketonuria (PKU) and Tay Sachs disease, to name a few.

Control Trial
As only catalase is used to assess the effect of pH on decomposition rate of hydrogen peroxide in this study, control trial is not required.

Random error
In Experiment 2c, the stopper popped off the conical flask at about the time interval of 30 seconds.This caused a slight drop in gas pressure, but the stopper was quickly connected to the conical flask.To minimise this error, the stopper should be pressed manually or clipped tightly to prevent leaking of oxygen gas.Additionally, although the chicken liver catalase was measured to a size of 1 cm 3 , measurement error may exist due to the texture and irregularity of the chicken liver.

Systematic Error
Systematic error can be minimised with using calibrated equipment that are reliable and functioning accurately including the chemistry sensor and stopwatch.

Limitation and improvement
The limitation of this study is only one trial is conducted.Future experiment can be improved by increasing sample size to repeat more trials to obtain average values, therefore reducing random errors.Furthermore, the experiment design can be improved by investigating more pH conditions to obtain a more accurate data to determine the optimum pH.

Conclusion
The experiment supports the hypotheses that the pH below and above the optimum pH decreases the rate of catalytic reaction of catalase breaking down hydrogen peroxide into oxygen gas and water, and that animal cells contain more catalase than plant cells.Chicken liver that contains higher catalase concentration per unit volume has a faster rate of reaction than potato.In this experiment, chicken liver catalase performs best at pH 6.5 while potato catalase works most effectively at pH 9.9.

Word Count
-1998 words -Headings, titles, figure captions, tables, references and logbook/journal are not included in the word count.

Background information/Introduction
What are enzymes?
• Enzymes are generally named for the reactions of what an enzyme does.One great example is DNA-polymerase which involves in DNA replication, it acts on DNA and synthesizes polymers of DNA.Generally, the suffix "ase" is usually used in most enzymes' names.• Enzymes are key players in a chemical reaction.
• Enzymes are biological catalysts that speed up the rate of reaction.
• Enzymes either split a substrate apart or combine them together.
• Induced fit model: The shape of the active site is very important to determine the function of the enzyme.

Figure 2a .
Figure 2a.Graph of potato catalase activity against pH

Figure 4 .
Figure 4. Graph of potato and chicken liver catalase activity against pH at 120 sec

Table 1 .
Stages of enzyme catalysis.

Equipment and Materials Materials Equipment Personal Protective Equipment
PASCO PASPORT Chemistry MultiMeasureSensor with pH probe and gas pressure tubing

Table 4 .
Experiment 2:The rate of reaction of chicken liver catalase decomposing hydrogen peroxide.

Duration Chicken Liver Catalase Rate of Reaction (Change in kPa)
Figure 3b.Graph of chicken liver catalase activity against pH at 120 sec

Table 5 .
The order of the rate of reaction measured in the change of gas pressure in kPa for potato catalase at 120 seconds.

Table 6 .
The order of the rate of reaction measured in the change of gas pressure in kPa for chicken liver catalase at 120 seconds.

Table 7 .
The rate of reaction of potato and chicken liver catalases decomposing hydrogen peroxide at 120 seconds.

Table 8 .
The optimum pH and greatest change in kPa for potato and chicken liver catalases.