Catalase activity in potato

Investigation into how pH levels affect Catalase activity in potato samples – (enzymes)
Introduction:
The aim of this investigation is to understand and monitor the effects pH levels have on catalase activity within controlled potato samples. Catalase is a type of enzyme that is found widespread among organisms that grow in the presence of oxygen (almost all living things) its specific use is to speed up the decomposition of Hydrogen peroxide (H2O2) within cells. Hydrogen peroxide is a bi-product of chemical processes such as respiration and is harmful if not removed or broken down into less harmful molecules. The enzyme (Catalase) acts as a catalyst, vastly accelerating this chemical reaction and allowing cells to function at a level where they can support life. Catalase
Hydrogen-peroxide Water + Oxygen
2H2O2 2H2O + O2

Enzymes are a protein made up of Amino Acids, they are essential to all living organisms as well as providing many commercial applications across multiple industries (including detergents, food and beverages, pharmaceuticals, biofuels, biodegradable plastics, leather and adhesive removal), within bio-detergents for example, the enzyme Protease is used to allow a deeper cleaning of textile materials at lower temperatures, while Pectinase (another form of enzyme found in plant cell walls) is used to partially digest fruit and vegetables in baby food (I). Each type of enzyme is unique in that it will only react with the substrate that complements the enzymes ‘Active Site’. The way the enzyme and substrate combine to react can be described as a ‘lock and key’ model, as the shape of each substrate (e.g. Hydrogen-peroxide), will only fit with the complementing enzyme (e.g. Catalase). (II) Having very specific and varying chemical reactions to make, each enzyme also exists in very different environments, for example Gastric lipase is an enzyme in the stomach of mammals that speeds up the digestion of foods, being in a very acidic environment their optimum pH range is 3-6 (III). When enzymes are exposed to pH levels above or below their optimum pH, the Active Site of the enzyme can become distorted or warped to the point where the complementary substrate can no longer fit in the active site, when this occurs the enzyme is known to have become ‘denatured’ (IV), this is very important to enzymes as they are reusable and become useless once denatured. Temperature also affects the activity of catalase as well as cause enzymes to become denatured, however this Investigation will monitor the activity of catalase across the pH spectrum and identify where activity is at its optimum, and at what pH the catalase enzyme becomes denatured (temperature will therefore be closely controlled).

Factors that might affect catalase activity: pH Levels
Temperature
Concentration of potato (catalase)
Potato type (source of catalase)
Concentration of Hydrogen-peroxide (Reactant)
Time (measure reaction rates)

Factor to monitor (independent variable) – pH: pH is one of the most important factors affecting catalase and all enzyme activity, by analysing reaction rates over a range of pH levels (pH3 – pH11), this investigation will show at what pH catalase activity will be at its optimum. As well as show at which levels the catalase enzyme becomes denatured (where environmental factors such as temperature or pH will irreparably damage the enzyme, essentially stopping the protein from functioning). A buffer solution is created for each of the pH levels included in the investigation.
Research and preliminary data obtained from a smaller experiment (figure 2 & table 2) utilising yeast as a source of catalase, suggest that enzymes will become denatured below pH3 and above pH11, for this reason and due to time restraints, the pH levels tested will be pH3, pH5, pH7, pH9 and pH11. This cross section of pH levels will provide tangible data as each step is equally separated, and within the environmental settings catalase can support reactions.

Strategy:

There are a number of ways that could be used to measure the rate of catalase activity:
Measuring the appearance of a product (O2) (V)
Measuring the rate of a disappearance of substrate (H2O2)
Measuring the pressure of the product as it appears (O2) (VI)
For this investigation I will be measuring the rate of the appearance of a product (in this case O2), the reason I chosen this method is that preliminary research suggest this is a wildly used and reliable study with the limitation of time and equipment available (V). O2 could also be measured by simply counting oxygen bubbles, however by measuring O2 in cm3 I can obtain more accurate and scientific results. The production of water (H2O) could also be monitored, however this would be difficult to measure as the amount of product is relatively small and translucent as with Hydrogen-peroxide.
Measuring the disappearance of substrate (H2O2) would be extremely difficult to measure by sight or with the equipment available to us, which also applies to measuring pressure.
Potato samples (catalase source) will be placed in a pH buffer solution before being exposed to the Hydrogen-peroxide (substrate), O2 production will then be measured in cm3 across a controlled period of time. The experiment will be conducted at a temperature of 25o as temperature has such a big effect on enzyme activity (as shown below). (VII)
Hypothesis:
Preliminary research (figure 2 & table 2) and my understanding of how enzymes function have led me to predict that when pH levels are altered, the rate of catalase activity will also change. My understanding of the subject tells me that each type of enzyme has a specific duty and will only react with its complementary substrate, research also identifies that the active site of enzymes can become denatured when exposed to extreme changes to pH or temperature outside to their natural environment. This shows me that catalase (as a type of enzyme) will have an optimum pH level for activity and that a pH level much higher or lower than this will cause the enzymes to become denatured, causing activity levels to fall and eventually stop as the substrate no longer fits in the active site. Based on this knowledge it’s therefore essential to maintain a steady even temperature throughout the experiment as well as control other variables such as sample size/surface area, and the time these samples are exposed to both the buffering solution and substrate (Hydrogen-peroxide, H2O2).
Preliminary research (figure 2 & table 2) has been conducted on yeast samples where 3 reading were taken at pH levels of 3, 7 and 11. Data readings suggest that catalase activity did not begin until after pH3 and peaked between pH7 & pH10. This evidence along with research from other sources supports my hypothesis that optimum activity will occur between pH8 and pH10. Catalase enzymes will become denatured and unreactive at and below pH3 and also denatured when reaching high pH alkali levels above pH10.

HHApparatus:
Apparatus
Image
Justification
Boiling Tube – (with delivery arm)

Contains the reactants (potato samples and Hydrogen-peroxide), and where the reaction is made. Arm is required for the Delivery tube allowing the passage of gas product (Oxygen)
Rubber Bung

Seals boiling tube from outside gasses, ensures all products from the reaction are unable to escape and follow their path to the measuring cylinder via the delivery tube
2 x Measuring Cylinder

Where product (Oxygen) is captured and used to measure the volume of oxygen (cm3), and thus the rate of the reaction. A further is used to measure accurate levels of substrate (Hydrogen-peroxide)
Delivery Tube

Connects boiling tube to the measuring cylinder, allowing the passage of gas product (Oxygen) for measurement
Beaker (filled with water)

Ensures the Measuring Cylinder is airtight allowing accurate readings of product (Oxygen) captured
Boiling Tube Rack

To safely hold Boiling Tube in position throughout the experiment
Digital Timer

Accurately measure how long potato sample is submerged in both buffer solution and Hydrogen-peroxide, ensuring a fair investigation
Thermometer

Ensure that temperature conditions remain stable, temperature could affect catalase activity and therefore should be controlled to ensure fair results
Scalpel

Precisely cut potato samples to required length, sample sizes should remain the same to ensure concentration of catalase within the experiment remain the same
Size 5 borer

Precisely measure potato sample sizes, ensuring catalase concentration remains constant throughout the experiment
White tile

White tiles provide a safer environment to cut samples of potato to required size and dimensions

Control Variables:
Control Variable
Why it needs controlling
How it’s controlled
Catalase concentration (potato sample)
Catalase concentration will affect the rate of reaction and ultimately the quantity of product (Oxygen) captured and measured.
All potato samples will be cut from using a size 5 borer and measured to 3cm in length. Each test will use 3 of these samples (total of 9cm), both to maximise the concentration of catalase and increase surface area of exposed potato flesh. All samples taken from the same potato species (Maris Piper)
Substrate concentration (Hydrogen-peroxide)
Hydrogen-peroxide (reactant) concentration will affect reaction rates being the substrate that enables catalase to drive reactions
Each test will contain 25cm3 of 6% Hydrogen-peroxide solution (H2O2)
Time measured
To ensure the investigation is fair, each sample needs the same amount of time exposed to both the buffering solution and the Hydrogen-peroxide (substrate), a greater exposure to either would alter the results of that test
For each test the 3 potato examples are submerged in buffer solution for precisely 1 minute. Once the first oxygen bubble is delivered to the measuring cylinder, volume is recorded after precisely 1 minute. Should no oxygen be measured after 3 minutes, record as 0.
Temperature
Temperature is a factor that greatly affects the activity levels of all enzymes, to ensure this doesn’t affect readings, temperature needs to remain constant
Temperature is controlled by conducting the investigation at a stable temperature of 25o

Risk Assessment:

Risk
What is the risk?
How to control
Risk level
Hydrogen-peroxide (H2O2)
Corrosive – Irritant to skin and eyes. Damage to clothing
Both goggles and lab coat to be worn at all times. The collection of the solution in small quantities
Medium
Glass
Breakages – Injuries – lacerations
Careful handling and the correct use of apparatus, keep glass away from table edge
Low
Scalpel
Injuries – lacerations
Careful handling, Only use with white tile
Low
Water & Solutions
Spill/Slip Hazard – Electric shock
Careful handling, ensuring all liquids are away from table edges and electrical sockets
Low

Equipment diagram:

Method:
1. Collect all equipment and clear all work surfaces.
2. Prepare boiling tube, measuring cylinder, delivery tube and all apparatus as shown in diagram above.
3. Using a cork borer cut 15 pieces of potato to 3cm long
4. Place 3 pieces of the potato samples into a pH 3 buffer for exactly 1 minute.
5. Place buffered potato samples into the boiling tube.
6. Measure out 25ml of 6% Hydrogen-peroxide (H2O2)
7. Add Hydrogen-peroxide and immediately place rubber bung to the top of the boiling tube, as soon as the first bubble of oxygen leaves the delivery tube, time for 1 minute. If no oxygen has been released after 3 minutes record the volume of 0cm3
8. After 1 minute from the time the first oxygen bubble has left the delivery tube, record the volume of oxygen captured within the measuring cylinder and record
9. Repeat steps 4-8 with the remaining buffer solutions (pH 5, 7, 9 & 11)
(To ensure accuracy and gather a greater field of data, repeat the experiment a further two times.)

Results:
Primary data: This table shows how much product (O2) was measured after one minute of measured production:
Table 1:

Test 1
Test 2
Test 3

pH Level
Cm3 of product (O2) collected
Cm3 of product (O2) collected
Cm3 of product (O2) collected
Mean (cm3) of product (O2)
3
0
0
0
0
5
2.0
2.0
2.0
2.0
7
2.5
2.75
3.0
2.75
9
4.5
4.0
3.5
4.0
11
3.0
3.25
4.0
3.42

(Figure 1) shows these results plotted onto a graph for analysis:

Preliminary data: This table provides the results from preliminary research where catalase activity was measured in yeast:
Table 2:

Test 1
Test 2
Test 3

pH Level
Cm3 of product (O2) collected
Cm3 of product (O2) collected
Cm3 of product (O2) collected
Mean (cm3) of product (O2)
3
0
0
0
0
7
0.5
2

0.85
11
1
3

1.55

(Figure 2) shows these results plotted onto a graph:
Secondary data (1):
The table below shows results obtained from a peer (Sean McDonnell) conducting the same experiment under the same conditions
Table 3:
(Figure 3) shows Sean McDonnells’ data plotted onto a graph:

Secondary data (2): The following table and supporting graph monitor O2 produced in a similar experiment (the only exception being that bubbles were counted rather than cm3 of O2 measured). This data has been collected from peers across the college.
Table 4:

Test 1
Test 2
Test 3

pH Level
Bubbles of product (O2) counted
Bubbles of product (O2) counted
Bubbles of product (O2) counted
Mean number of bubbles product (O2) counted
3
0.0
0.0
0.0
0.0
5
4.0
7.0
5.5
5.5
7
10.0
15.0
11.0
12.0
9
8.0
10
8.0
8.6
11
0.0
1.0
0.2
0.4

(Figure 4) – Shows this data plotted onto a graph

Secondary data (Temperature): The following article is added as a supporting article to demonstrate the effect temperature has on catalase activity. http://practicalbio.blogspot.co.uk/2012/03/easy-enzyme-experiment-potato-catalase.html Results analysis:
Primary results collected along with supporting evidence confirm that pH levels are extremely important to the rates catalase/enzymes can react. For example, every data piece taken has suggested no activity at pH3, this means the enzymes have become denatured and can no longer drive reaction rates as the substrate (in this case H2O2 - Hydrogen-peroxide) no longer fits into the active site of the enzyme (catalase) rendering it useless. There are however some discrepancies in the data. Both my preliminary data and primary experiment have provided more volume of product (O2 – oxygen) at pH11 than expected, which is confirmed by the secondary data showing a much lower production of the gas at this high alkali level.
The full set of results also provides questions on where the enzymes are reacting at their optimum level. Although every graph shows the same trends in terms of ‘limited to no’ activity at pH3, and correlate in shape to where reaction levels begin to drop, actually determining at what pH the catalase enzyme is at its optimum is not so clear. Table 4 suggests pH7, Table 3 suggests pH9 whilst Table 2 suggest a pH of 11 which is a very broad spectrum almost taking up half of the full pH spectrum. The full spread of data combined does give me confidence in my prediction, even though the investigation could have provided more consistent results.

Evaluation:
Contamination in the primary experiment – conducting every test of the pH at the same level rather than doing the series and repeating
Temperature – temperature rates where different as over 2 day – due to previous remark pH 11 was taken in different conditions than the previous pH levels
Eye level – reading of product level – inaccurate in the grand scheme of things – more precise measurement
Didn’t have any unforeseen issues with equipment availability
Time was an issue and
Bung correct
Keep talking problems
2 different potato
Should have done
Measure with pressure sensor or gas reader – more accurate reading
Control temp better – all on one day
Run experiment in series
Use fresh boiling tube and apparatus for each test
Samples for more time?
Bigger samples – bigger scale experiment
Same potato

References:

(I) http://www.s-cool.co.uk/a-level/biology/biological-molecules-and-enzymes/revise-it/industrial-enzymes
(II) http://wps.prenhall.com/wps/media/objects/3312/3391801/blb1406.html
(III) http://en.wikipedia.org/wiki/Digestive_enzyme
(IV) http://www.bbc.co.uk/schools/gcsebitesize/science/add_edexcel/cells/enzymesrev3.shtml
(V) http://www.rsc.org/Education/EiC/issues/2007Jan/InvestigatingActivationEnergies.asp
(VI) http://www2.vernier.com/sample_labs/BIO-A-02B-COMP-enzyme_action.pdf
(VII) http://www.bbc.co.uk/schools/gcsebitesize/science/add_aqa/proteins/proteinsrev3.shtml