How do variations in affect the reaction time of the decomposition of hydrogen peroxide, involving catalase (enzyme)?

Name
Simran Bhalla

Design
Using your knowledge of factors that affect enzyme activity, you are asked to design an investigation to look at the effect of one chosen factor on the enzyme catalyse. Catalase is an enzyme that hydrolyses the breakdown of hydrogen peroxide into water and oxygen.

Research Question
How does the pH (5, 6, 7, 8 and 9) affect the reaction time of the decomposition of hydrogen peroxide, involving catalase (an enzyme)?

Background Theory
Enzymes are biological catalysts that speed by metabolic reactions in the body. Some of these reactions produce toxic by-products1, which must be quickly degraded or converted. An example of an enzyme is catalase, responsible for the decomposition of hydrogen peroxide, a toxic substance to cells. This enzyme is found in the peroxisome microbody2, in the cells of eukaryotic organisms. These form hydrogen peroxide as a by-product of anaerobic respiration3. Catalyse has amongst the largest turnover numbers4 of all human enzymes, as one of its molecules can convert many millions of hydrogen peroxide particles into water and oxygen each second. It can be found in beef liver, potatoes, wheat sprouts and carrots. Chemical Reaction of the decomposition of hydrogen peroxide using catalyse

Substrates are the chemicals that attach to the enzymes active site with induced fit. The combining of the enzyme and substrate, a reversible reaction, requires the use of ATP. However, it should be taken into consideration that when enzymes are used in the human body, it is vital that they work under conditions optimal for them, so they can work to the best of their ability. These include: substrate concentration, temperature, pH levels, presence of activators etc.

Science Geek. n.d.. Effect of Catalase on Hydrogen Peroxide. [online] Available at: http://www.sciencegeek.net/Biology/biopdfs/Lab_Catalase.pdf [Accessed: 20 Jun 2013]. 2 Parish, R. 1975. The isolation and characterization of peroxisomes (microbodies) from baker's yeast, Saccharomyces cerevisiae - Springer. [online] Available at: http://link.springer.com/article/10.1007%2FBF00447136#page-1 [Accessed: 20 Jun 2013]. 3 Learn Premium UK. n.d.. The effect of pH on catalase activity. [online] Available at: http://www.ngflcymru.org.uk/vtc/phase1/wales/aslevel/biology/molecules/enzymes/factorsaffectingenzymefunctioning/Expe rimenttheeffectofpHoncatalaseactivity/ [Accessed: 20 Jun 2013] 4 En.wikipedia.org. 1900. Catalase - Wikipedia, the free encyclopedia. [online] Available at: http://en.wikipedia.org/wiki/Catalase [Accessed: 20 Jun 2013].

For most enzymes, including catalase, the optimal temperature is around 37oC, while the pH levels vary from 6.8 to 7.05. If the pH of the enzymes environment becomes more acidic or basic, the active site is forced to change its shape and resultant, the substrate cant attach. Thus, the enzyme is denatured. Effect of pH on enzyme activity shown on a graph

Hypothesis
The reaction time of the decomposition of hydrogen peroxide (involving catalase) will increase at pHs above and below the optimal pH of 7 of the enzyme, catalase. The lowest reaction time will be noted by the optimal pH of 7. This is because for most enzymes, the ideal pH at which enzyme activity is fasters is pH 7, and when not at that level, the efficiency of the enzyme activity can be sufficiently reduced. Moreover, in greatly acidic or basic environments, the enzyme can denature.

Variables
1) Independent Variable pH of the reaction environment (buffer solution)- pH 5, 6, 7, 8, 9 2) Dependent Variable Reaction time of the decomposition of hydrogen peroxide (involving catalase) measured by the time taken for the oxygen bubble production to conclude.

US National Library of Medicine. 1928. THE DECOMPOSITION OF HYDROGEN PEROXIDE BY LIVER CATALASE. [online] Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2140981/ [Accessed: 20 Jun 2013].

Controlled Variables
Variable Controlled Why it needs to be controlled? How will it be controlled?

Amount of enzyme catalase will be the same

Temperature of the environment will approximately remain the same

To ensure that a fair test is being conducted- having a greater amount of catalyse will most likely result in that particular test having a lower reaction time. Temperature is a factor, like pH, which has a direct effect on the activity of the enzyme. Therefore, to ensure a fair test, this has to be controlled.

The pieces of potato (the source of the catalase in the experiment) will be 1cm2 cubes in size.

Amount of hydrogen peroxide will be the same Human error using a stopwatch

Substrate concentration

To ensure that a fair test is being conducted- having more H2O2 in one test will most likely result in a longer reaction time. Thus, accuracy will be affected. There is a reasonable scope of human error or bias when using a stopwatch, which can affect the precision of the experiment (and accuracy to some extent). Substrate concentration is factor, like pH, which has a direct effect on the activity of the enzyme. A higher concentration, can lead to a lower reaction time.

The air-conditioning of the lab will remain constantly at 24oC and the area will be under equal amounts of artificial light (i.e. light tubes) in addition to no direct sunlight. A thermometer will be used to ensure that the temperature remains between 22-26oC 3ml of hydrogen peroxide will be used in each test, added via a syringe. The same person will time the experiment each time.

3% hydrogen peroxide solution will be used in this experiment.

Materials and Apparatus

25 1cm3 cubes of potato [1 cube for each test] 75ml of 3% hydrogen peroxide solution [3ml for each test] Disodium Phosphate (Na2PO4) buffer solution a. 1250ml pH 5 [250ml for each test] b. 1250ml pH 6 [250ml for each test] c. 1250ml pH 7 [250ml for each test] d. 1250ml pH 8 [250ml for each test] e. 1250ml pH 9 [250ml for each test] 6250ml of distilled water 1 Conical Flask ( 5ml) 1 beaker, 250ml ( 5ml) Measuring cylinder, 100ml ( 2.5 ml) Delivery Tube Thermometer 1 Sterile syringe, 5ml ( 0.5 ml) 1 Rubber stopper, with 2 holes Knife iPhone Stopwatch Application ( 1.3 secs) Chopping Board Ruler

 Writing apparatus

Risk Assessment
Hydrogen Peroxide can be toxic, but the 3% Hydrogen Peroxide solution being utilised in this practical has a relatively low hazard. However, if the chemical makes contact with skin or eyes, irritation can be initiated and it is then recommended to flood the affected area with water. Ingestion of this substances is strongly discourages. Eye protection should be worn at all times when using hydrogen peroxide.

Method Visual Set-up

Step-by-Step Instructions
1) Ensure that the air conditioning is at 24oC to keep the temperature constant. 2) Set-up the experiment area as shown in the diagram above. Place a potato cube in the conical flask and add 250ml of pH 5 buffer solution. On the top of the conical flask, put a rubber stop connected to a syringe and delivery tube, which ends at a beaker containing distilled water. 3) Fill up the syringe with hydrogen peroxide. Inject it into the conical flask (as shown in the diagram) and simultaneously start the timer. Note the formation of oxygen bubbles inside the inverted measuring cylinder. Once this process ends, stop the timer. 4) Record the reaction time and qualitative observations. Rinse the conical flask and delivery tube, and empty/ refill the beaker. 5) Repeat steps 1-3 for each of the other pH buffer solutions (pH 6, 7, 8 and 9) 6) Repeat steps 1-4 four more times, to maintain the reliability of the results. 7) Calculate the average reaction time for each different pH and compare the final figures to determine what independent variable had the lowest average reaction time. The formula for calculating the average is:

Blank Table of Results

pH Level 5 6 7 8 9 Trial 1 Reaction Time (seconds)( 1.3 secs) Trial 2 Trial 3 Trial 4 Trial 5