Liver And Hydrogen Peroxide Lab Report

Biology investigation: Liver and hydrogen peroxide
By Cuong Tran
Abstract: The purpose of this investigation was to determine the optimum temperature for enzyme activity. The stated hypothesis was that 37oC would be the optimum temperature for liver enzymes to break down hydrogen peroxide into water and oxygen. The dependent variable was to measure the amount of foam being produced, while testing 4 different temperatures with 3 repetitions each. Out of the 12 trials only 2 outliers were found and an inclusive overlook of random and systematic errors were explode to determine the possible causes. The final results support the hypothesis that 37oC was indeed the optimum temperature for enzyme activity, producing the greatest volume of foam
at 44mL.

Introduction:
An enzyme- catalase chemical reaction is a metabolic pathway responsible for metabolising chemicals in both animals and plants (Greig, Oct 2002). In mammalian this reaction takes place in the liver for detoxification, were toxic substances are broken down in the body into harmless chemicals for the body to sustain (Victoria, Australian government, 2011). The reaction involves a protein enzyme called catalase a biological catalyst that speed up reactions and lowers the activation energy by binding to a biotic or abiotic material named a substrate (Greig, Oct 2002). The binding process or lock and key model is when a substrate binds with the active site of the enzyme, it forms an enzyme-substrate complex, usually held by weak bonds. (Greig, Oct 2002) The weak bonds place stress on the substrate, which is the initial energy necessary to start the reaction or activation energy (Greig, Oct 2002). It breaks down hydrogen peroxide into harmless substances water and oxygen (figure 1). The products of the substrate then depart from the active site of the enzyme, leaving it free to catalyse another substrate as the cycle begins again. (Greig, Oct 2002)

Full balanced equation:
2H2O2 (aq) - 2H2O(aq) + O2 (g)
Hydrogen peroxide  water + oxygen
Oxidation: H2O2 --> O2 + 2H+ + 2e-
Reduction: H2O2 --> 2H+ + 2e- + 2H2O
(figure 1)

H2O2 is naturally produced in the body as a signal for white blood cells to attack, pathogens and bacteria. Although a build up of H2O2 (reactant) in the body can also be lethal, killing cells and causing liver damage at extreme cases. All mammalians produce H2O2 for the same purpose and all contains a liver enzyme called a catalase that is responsible for decomposition of H2O2. The velocity of the decomposition of H2O2 can depended on veriest factors, one being temperature. In this investigation the by-product oxygen used to measure the rate of reaction between the catalase and H2O­2. Oxygen is produced in the form of a gas, combined with detergent the bubbles of oxygen produce foam. The amount of oxygen produce can depend on different factors, one important physical factor being temperature (Greig, Oct 2002). Because this reaction takes place in all living organism, mammalian have an optimum temperature of 35-40oC for enzyme activity (Greig, Oct 2002). The optimum temperature is a certain point were the collision of the substrate molecules is more frequent, thus is most likely to interact with the active site of the enzyme produce more of the by-product (Greig, Oct 2002). When the temperature exceeds the optimum, the enzyme protein denatures, destabilizing the active site making it hard for the substrate to bind with the enzyme (Greig, Oct 2002). At low temperatures the rate of reaction is much slower due to less collision between the substrate and enzyme, thus fewer by-products. (Greig, Oct 2002)

Aim: To find the optimum temperature for enzyme and substrate reaction between hydrogen peroxide and the liver catalase.

Hypothesis: Because the human optimum temperatures for hydrogen peroxide to be broken down by liver enzymes is 35-40oC. It is determined that the optimum for enzyme activity will be 37oC.

Variables:
Independent: Changing the temperature of water and hydrogen peroxide and liver
Dependent: Measuring the volume of foam being produce in each repetition
Controlled: Volume of liver (1 capsule), concentration of hydrogen peroxide (6%) and volume (5mL), (1ml) of detergent. 27oC room temperature, same apparatus. Timing 1 minute 30 seconds, and keep surface area to volume ratio even throughout each trial

Safety :
Personal protection equipment
Must always wear PPE during the experiment this includes gloves, lab coat, glasses and fully covered enclose shoes
Safety Procedure:( Call of a teacher for any assists)
-Broken glassware- If broken glass is apparent make sure to alert others and clean up with dust pan and dispose in sharp bin.
-Hydrogen peroxide- If direct contact with hydrogen peroxide occurs, eyes or skin, an emergency shower and eye wash is located near the exit door. Making sure to wash from 5 to 10 minutes or until hydrogen peroxide has completely washed away.
-Burns- If burnt quickly apply ice pack to reduce swelling

Apparatus
-50mls of 6% hydrogen peroxide -4 100ml measuring cylinders
-12 of mammalian liver capsules -PPE (Personal Protection equipment)
-3 syringes - 3 thermometer
-stopwatch - kettle
-ice - 3 beakers
- 12mL of detergent

Method:
Preparing the liver
1. Cut up small chunks of liver using a knife and place them into a blender with a small amount of water.
2. Blend the liver until it is consistent and all the liver has been pureed.
3. Using a syringe, insert the blended liver into the capsules making sure each capsule has the same volume of liver.
4. Once all the liver has been inserted into the capsules, place them into the centrifuge, then let it spin for 4-6 minutes or until there is distinct line between the impure and pure liver occurs.

Procedures:
1. Collect all materials needed for experiment, making sure all glass are clean. Wash with tap water then distilled and rinse.
2. Set up data table to record results for 4 of the temperatures and 3 repetitions for each.
3. For 10oC, place small amounts of part ice and water into a beaker (water bath) and wait till it has reached 10oC. Once reached keep temperature constant by adding in salt.
4. Add in 1mL of detergent and 5mL of hydrogen peroxide into measuring cylinder, then place in the beaker measure the temperature IN the test tube also for 10oC
5. Once both the water bath and measuring cylinder are at set temperature, add in liver with a syringe, making sure only to collect the pure layer.
6. Once collected add liver to test tube containing the hydrogen peroxide and detergent, carefully insert the liver to the bottom on the measuring cylinder, making sure no liver is left on the sides. Once placed start the timer for 1 minute 30 seconds.
7. Once time is up, make sure that when recording the final volume, for your eyes is levelled and in line with the measuring cylinder.
8. Repeats steps 1 to 7, 2 more times for 10oC. Each set temperature should have 3 repetitions As well for 37oc, 50oc and 70oC, instead using the kettle to meet the desired temperature.

Results table: The affect of temperature on enzymes activity (figure 2)
Temperature (oC)
1st (mL)
2nd (mL)
3rd (mL)
Averages (mL)
10
20.00
20.00
20.00
20.00
37
44.00
33.10
44.00
40.40
50
32.20
25.00
25.00
27.40
70
15.00
15.00
15.00
15.00

Graphs: The volume of foam been produced at different temperatures within 1 minute 30 seconds.

Figure 2 shows the raw data of the 3 trials for 4 different temperature in the experiment, with the outliers highlighted in yellow and red.
The bar graph above represents a visual overall look of how temperature affects the reaction rate. The dependent variable was to measure the volume of foam been produced at each temperature with 3 repetitions. The graph shows that 37oC was the optimum temperature and producing the greatest height of reaction 44mL. However, as the temperature continues to increase the enzymes begin to denature resulting in the lowest recorded volume of 15mL at 70oC. Although the results do support the hypothesis for 37oC the standard deviation was 6.29, while 50oC had a standard deviation of 4.15. Out of the 12 trials 2 outliers were apparent 2nd trial at 37oC and 1st trial at 50oC which may have been caused by random and systematic errors throughout the
experiment.

Discussion:
A possible random error that may have caused these 2 outliers is sudden temperature fluctuation. This random error may have caused a drop or rise of temperature in the test tube or in the water bath. Either slowly down the collusion between the enzymes and substrates or possibly denaturing the enzyme altering the active site (Lower, 2011). Keeping the set temperature constant was a problem, while testing each temperature each trial went for 1 minute and 30 seconds. In that time the temperature of the water bath or test tube may have risen or dropped, changing the kinetic energy created by the collision of enzymes and substrates. If the kinetic energy isn’t kept constant throughout the duration, as the temperature lowers, the collision become less and less frequent resulting in a lower reaction rate. As the reaction rate declines the reaction rate is inconsistent, as the first 30 seconds of the trial may have produced more of O2 and H2O then the last 30 seconds (Lower, 2011). A sudden rise in temperature however can result in the enzyme being denatured altering the active site. Without the collision between the two molecules there is no energy supplied to break the chemical bonds of the substrate.

Hot and cold pockets may have also affected the sudden temperature change. Because the detergent and hydrogen peroxide were added into the test tube at the same time till set temperature, the liver was added last which sat at room temperature (27oC), which explains why trial 1 at 50oC only produced 32mL of foam. Which is relatively the same compared to trial 2 at 37oC. The liver may have created a cold pocket in the test tube which allowed the set temperature of 50oC to fluctuate, lowering the reaction rate. This problem may have also occurred to trial 2 at 37oC, lowering the temperature, the collision become less frequent, and creating less energy to start the activation energy. Without ignition the activation energy the catalase aren’t able to distort the H2O2 into its products, producing less O2. (Ladiges, 2008)

The poor handling of the syringes may of also affected the amount of catalase in the test tube during the reaction. Because each individual reactant molecule have different energies. In order for the substrate to decompose itself the two molecules must possess more than the minimal level of energy necessary to break the bonds at the instant they collide. Those molecules with less than this amount of energy will not react at all, the energy required to initiate the reaction is the activation energy. With fewer catalase the activation energy for each of these molecules are not active, which could possibly means half of the substrates didn't have the required energy to break the bonds of H2O2. (Ladiges, 2008)

The different concentration of liver catalase can also be an effective error by having the unknown amount catalyst in each liver capsule. (Lower, 2011). Each section of liver may have differed in concentration of enzymes, meaning each liver capsule may have had more or less enzymes for the substrates to bind too. Creating an inconsistent reaction rate for possibly all the trials. (Ladiges, 2008)

Another random error is the surface area to volume ration. While the liver was in the centrifuge to create a pure layer of liver on the surface, leaving the impurity on the bottom, the impurity may have been inserted into the syringe with the pure liver and used in the trials. The impurity mainly resolves around inconsistent surface area, affecting the number of interactions between the enzyme and substrate causing the reaction rate to lower. The more even the surface area to volume ratio, the more consistent the reaction rate is.

Systematic errors also occurred during this experiment that affected these outliers, or affected all the results, bringing it further apart from the true value. One possible systematic that may have affected the results would be the concentration of H2O2. Because the concentration of H2O2 on the bottle was 6%, this may of have dropped slightly during the experiment. H2O2 when exposed to the air naturally oxidises and reduces to water and oxygen gas (fig 1), therefore reducing the concentration of H2O2 molecules in the solution over time. Possibly making the concentration of H2O2 less than 6%. Whilst the H2O2 was in the conical flask, it may have oxidised during the experiment making the concentration less and less every 2 or 3 trial. The temperature for the trial may of been exact, although if the concentration had lessen this may of caused the collision to lower. (Ladiges, 2008)

Parallax can also be a random error, either miss reading the final volume or wrongly determining the peak of the foam because of its apparent position (Princeton University, 2012). This error may have happened because of how poorly the syringes were handed while inserting the liver, detergent and H2O2 in the measuring cylinder. Small samples of each may of have remained on the side of the cylinder, resulting in the reaction taking place on the side and not at the bottom of the cylinder. Because the reaction was taking place in two different areas in the cylinder, this made it difficult to determine the true peak of the foam.

The reliability of this experiment is varied, because of these random and systematic errors. If this experiment were to be done again, either 2-4 repetitions, the results may be inconsistent over time (Goodfriend, 2014). However if this experiment were to be tested in another lab or area, these results also wouldn't be reliable across samples, because of new variables that may alter the experiment. It could also be said that these results wouldn't reliable across experimenters as well (Goodfriend, 2014). It is possible tho to minimize these errors, which could bring the precision closer and accuracy to the true value.

The validity of this experiment is the purpose of why this investigation was conducted. The internal validity of this experiment is that it does measure the cause and effect, of how temperature affects the reaction rate of enzymes (Goodfriend, 2014). The external validity, how these findings are true to other experiments being conducted, these results do support the hypothesis, and research has confirmed that 37oC is the optimum temperature for catalase reaction in the human body (Goodfriend, 2014). The ecological validity, the reason why this experiment was conducted and how it relates to everyday people (Goodfriend, 2014). Because the optimum temperature for enzyme activity is 37oC, the results confirm this and support the hypothesis that in order for the body of mammalian to break down excessive harmful substance such as H2O2. The optimum temperature must be 37oC.

Conclusion:
This investigation was conducted to determine the optimum temperature for enzyme activity. By using 4 different temperatures the results conc (Ladiges, 2008)lude that 37oC was the optimum temperature for hydrogen peroxide to be broken down as it produced the greatest amount of oxygen. Thus this supports the hypothesis that in order for the liver in mammalian to carry out detoxification at its optimum rate, the internal body temperature must be 37oC.

Evaluation/improvement:
While the results obtained from this investigation do support the hypothesis, outliers were still found throughout the experiment, which affects the precision of the data.
This investigation could have been improved if more trials were tested, around 4 to 6, this would ensure a better overall average of each temperature and standard deviation to the true value. Adding in 1 or 2 more set temperatures to be tested such as 20oC and 40oC would also give an overall overlook of the reaction rate of enzymes. Where reaction of enzyme activity is at its peak and then when it begins to denature. Another improvement would be to concentrate on one water bath at a time. While testing the reaction rate in one water bath the other may have when left unattended either cooled off or warmed up. Because some of the data was varied compared to other groups, there may of have been an error with the liver itself. While the pure samples of liver were the same surface area, not all pieces were blended from the same section of the
liver.