temperature on the activity of liver

Introduction:

Catalase is a common enzyme found in living and it acts as a protective mechanism for delicate biochemical machinery of cells. The enzyme catalyzes the exothermic decomposition of hydrogen peroxide to water and oxygen.

2 H2O2 → 2 H2O + O2

Hydrogen Peroxide is a by product produced by many living organisms during the process of metabolism. Hydrogen Peroxide is a very toxic substance (a power oxidizing agent) to cells and must be broken down in order to protect the cells from subsequent damage.

Aim:

The aim of the experiment is to investigate the effect of varying temperature has on the rate of enzyme catalyzed reaction. The focused reaction is the decomposition of hydrogen peroxide with the enzyme catalyze. The presence of catalase can be demonstrated by dropping a small piece of fresh liver tissue into dilute hydrogen peroxide solution. In this experiment, pieces of liver tissue will be put into different temperatures of water for 5 minutes. After that the liver tissues will be placed into separate solutions of hydrogen peroxide and the amount of oxygen gas produced in a minute will be measured using a gas syringe.

Hypothesis:

Temperature is a measurement of the degree of hotness or coldness of a body or environment. More specifically, it is a measure of kinetic energy in a sample of matter. On a molecular level, temperature is the result of motion of particles which make up a substance. As the temperature increases, the motion also increases. The motion may be due to external energy applied to the particle of internal energy from the vibration of the particle. As temperature is increased, molecules have increased active energy and reactions between them and the probability that the particles will collide with each other will also be greater, this increasing the rate of reaction. In chemical reactions, for every 10°C rise in temperature, the rate of reaction approximately doubles. This property is known as the temperature coefficient of a chemical reaction. However in a enzyme catalyzed reaction the effect of temperature is more complex, for proteins change shape by heat. There are many factors that can affect the structure of a protein such as temperature and ph. When a protein is exposed to heat, it causes the atoms to vibrate violently, breaking and disturbing bonds within the protein, therefore changing the chemical characteristics of the protein. I hypothesize that as the temperature of the water bath that the liver tissue is exposed increases; the amount of oxygen gas liberated will also increase up. I believe that there will be an optimum temperature for the enzyme and going pass the optimum level will cause a drastic decrease in enzyme activity (less oxygen gas will be produced). Since catalase is found in almost all living things, including humans, I predict that the optimum temperature for catalase will be

Variables:

Independent Variable

Temperature of water bath liver tissue is placed In (°C)

Dependant Variable

Volume of oxygen produced in a minute (ml/min)

Controlled Variable

Concentration of the Hydrogen Peroxide

Volume of Hydrogen Peroxide

Mass of liver tissue

The concentration of hydrogen peroxide must be kept constant because according to the Collision Theory proposed by Max Trautz and William Lewis in 1916 and 1918, increasing the concentration, increases the chances of particles hitting each other. The volume of hydrogen peroxide should also be kept constant. Increasing the volume of hydrogen peroxide increase the substrate concentration and thus increasing the rate of reaction. Finally the mass of liver tissue should also be kept constant to try control the amount of enzyme molecules present. Increasing the number of enzymes means there are more active sites present and substrate molecules do not have to “queue up” for access to an active site. Ultimately increasing enzyme concentration can also result in an increase in rate of reaction therefore the mass of the liver tissue should also be controlled.

Equipment:

Safety Note:

• Eye protection should be worn at all times
• If liquid gets into eye, flood the eye with a gentle running tap for 10 minutes and seek medical attention
• If hydrogen peroxide is spilt in the lab, cover it with mineral absorbent. Dilute with water and wash liquid.
• Hydrogen peroxide should be stored in a dark brown bottle and care must be taken when removing the cap as it is possible that pressure may have built up inside it.

Method:

1. Draw up a suitable table or tables to record the results.
2. Carefully cut 7 pieces of cow liver tissue using a knife and a cutting mat.
3. Weigh each piece of liver tissue carefully on the electric balance. Make sure each liver tissue weighs roughly around 0.5 grams.
4. Place each liver tissue into a separate boiling tube and add 40ml of deionized water to each boiling tube once the liver tissue is situated at the bottom of the boiling tube.
5. Place the heating mat on the table with the tripod on top of the heating mat. Gently place the gauze on the tripod. Once this is done, place the beaker on the tripod and slowly heat up the water with a Bunsen burner. Place a boiling tube with a liver tissue sample into the water and put a thermometer in the tube. Heat the beaker until liver sample solution reaches 70°C. Measure temperature of water with a thermometer.
6. After that, carefully measure out 100ml of hydrogen peroxide with a measuring cylinder and transfer the solution to a 250ml conical flask.
7. Connect one end of the gas delivery tube to the gas syringe and the other to the conical flask
8. Remove the liver tissue from the boiling tube with a pair of tweezers and place it into the conical flask with the hydrogen peroxide.
9. Quickly cork the conical flask once the liver tissue is dropped into the solution of hydrogen peroxide. Beginning timing the time once the liver tissue touches the hydrogen peroxide solution.
10. Stop the stop watch after 1 minute and record the amount of gas produced. Read off the gas syringe.
11. When the reading is taken, remove the cork and dispose of the hydrogen peroxide in the chemical waste container.
12. Repeat the above steps until data points from 10°C to 70°C are recorded.. For readings below 30°C, cool the liver tissue sample with an ice bath.

Diagram:

Results:

Table of Results

Table 1.0 – Raw Data

Table 1.1 – Qualitative Observations

Graph 1.0 – Temperature and the Amount of Oxygen Liberated from Liver Tissue Sample

Graph 1.0 – The graph above shows the relationship between the temperature of the water bath the liver tissue sample was put it and the amount of oxygen gas liberated from the sample after dropping it in dilute hydrogen peroxide in 1 minute. The graph clearing shows that as the temperature increases, the amount of gas also increases up to 60°C. From 60°C onwards, the amount of oxygen gas produced decreases drastically and there is a downward slope of the curve.

Discussion:

From the data obtained, there is an increase of oxygen produced as the temperature of the water bath increases. This trend however only applies to the data points from 20-60°C. At 70°C however, there is a significant drop in the amount of oxygen gas produced and at 80°C, the amount of oxygen gas produced is less than 5ml. From the graph, the relationship is clearly represented. Up to about 60°C the amount of oxygen gas produced increases and ten-degree rise in temperature is accompanied by 6-7ml increases in oxygen gas produced. The amount of oxygen gas produced decrease at high temperatures as shown from 70-80°C. So as the temperature rises, the amount of enzyme progressively decreases and the amount of gas produced is less. As a result of these two effects of heat on enzyme, there is an apparent temperature for an enzyme. Using the graph, the optimum temperature of catalase is approximately at 60°C.

The properties of a protein greatly depends on its three dimensional shape of the molecule. Exposure to heat causes the atoms to vibrate violently and this can cause bonds within the protein between different amino acid to break, resulting in a loss of the proteins biological properties. This is known as denaturation of a protein. Heating causes a protein’s biological properties to change such as optical rotation, shape of active site and bonding. The active site of the enzyme is what defines the enzyme. If the active site changes, the substrate molecules will no longer fit the active site of the enzyme. This is shown in the diagram below.

A protein’s stability depends primary on the hydrophobic effects and to a lesser extent, by the interactions between polar residues and by other types of bonding. There are four levels of protein structure each playing a significant in the stability of the protein. The primary structure of a protein is the sequence of amino acid residues attached by peptide linkages. Proteins differ in the variety, number and the order if their constituent amino acids. Changes is just one specific amino acid in the sequence of a protein can alter a protein’s property. The secondary structure of a protein develops when parts of the polypeptide chain take up a particular shape. The most common shapes are either coiling to form α helixes or into β sheets. The tertiary structure of a protein is the distinctive and precise structure, unique to that specific protein. This is usually the result of further folding and interactions within the molecule. The shape is held together permanently by four different types of bonds: hydrogen bonding between hydroxyl and ketone functional groups, Van der Waals forces between neighboring atoms, disulphide bonds between two cysteine side chains and ionic bonds between oppositely charged ions. The stability of a protein is dependant of the balance of these three structures. Altering the balance of forces that maintains the native conformation of the protein will lead to denaturation.

Conclusion:

The hypothesis put forth before the experiment was correct. As the temperature of the water bath that the liver tissue is exposed increases; the amount of oxygen gas liberated will also increase up to a certain level. That level would be the optimum temperature of the enzyme and the optimum temperature of catalase deduced from the experiment is approximately 60°C. Pass the optimum temperature, the amount of oxygen gas produced drastically decreases as the enzyme is starting to denature. At 80°C, the enzyme is almost fully denatured and only a small amount of oxygen gas is produced. The literature value for the optimum temperature of catalase in human is about 37°C which is the body temperature of a typical human being. The liver sample was taken from a cow and both cows and humans are able to regulate their own temperature. From this we can deduce that the optimum temperature of catalase for a cow should be roughly similar to a human’s and somewhere around 37 -40°C. The optimum temperature obtained from the experiment is 60°C, this suggests that there may have been deviations or variations within the data.

Limitation to Experiment:

There are several limitations to our experiment to the experiment. One of the main limitations was controlling the temperature of the water baths. The temperature began to drop gradually once the boil tubes were placed into the beaker. This may have caused deviation and variations within the data. Another limitation was that the experiment was only repeated 3 times and there may still be room for anomalies and errors.

Evaluation:

As long as the temperature of the water baths were kept constant and the amount of gas produced was measured and read correctly, a good approximation of the optimum temperature of catalase in a cow can be found. However there are several key improvements that can be made. When the liver tissues were cut a lot was wasted and some of it got stuck to the spatula and to the top of the test tube. To compact the problem in the near future, the liver tissues stuck onto spatula could be washed off using a tiny amount of water or shook gently to try remove some of the liver tissue. The method for collected the oxygen gas could also have been improved. Some gas was lost due during the time it took me to insert the bung into the test tube. To minimize the amount of gas lost, have a partner put the bung on for you while you drop the liver tissue into the measuring cylinder. The temperature of the water baths started to drop after a few minutes and this may have lead to some slight deviation within the data. In future make sure that the temperature of the water bath does not change too significantly. A slight modification would be to cover the beakers with a lid to prevent air from escaping. Another problem that arose during the experiment was keeping the temperatures of the water baths constant during tests and between trials. The problem was that different temperatures of liver samples were tested and it wouldn’t be feasible to use a electronically controlled water bath to perform each test. The result was that a Bunsen burner was used but this creates several problems. It was extremely difficult to get exact temperatures and maintain them throughout the experiment. In future, the use of a heating plate would be more practical so as to prevent drastic changes in temperatures. To improve the experiment it may be better to use a solution of catalase rather than a liver as a source of catalase. By using a solution, the concentration and volume could be controlled and would be the whole experiment into a fairer test. Overall the results obtained have helped support the hypothesis put forth before the experiment. Finally the experiment could have been repeated a few more times so that a better average could be drawn from the data, resulting in a more reliable and accurate conclusion.

References

Clegg, C. J. (2007). Biology for the IB Diploma. London: Hodder Murray.

Ophardt, C. E. (2003). Denaturation of Proteins. Retrieved January 3, 2009, from Elnhurst College

Web site: http://www.elmhurst.edu/~chm/vchembook/568denaturation.html

Roberts, M. B. (1986). Biology a functional approach (4th ed.). Canada: Nelson.

Voet, D. J., Voet, J. G., & Pratt, C. W. (2008). Principles of Biochemistry. NJ: Jon Wiley & Sons,

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