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Introduction:

Catalase is a common enzyme found in life and it acts as a protective mechanism for delicate biochemical machinery of cells. The enzyme catalyzes the exothermal decomposition of H peroxide to H2O and O.

2 H2O2 a†’ 2 H2O + O2

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Hydrogen Peroxide is a by merchandise produced by many populating beings during the procedure of metamorphosis. Hydrogen Peroxide is a really toxic substance ( a power oxidising agent ) to cells and must be broken down in order to protect the cells from subsequent harm.

Purpose:

The purpose of the experiment is to look into the consequence of changing temperature has on the rate of enzyme catalyzed reaction. The focussed reaction is the decomposition of H peroxide with the enzyme catalyze. The presence of catalase can be demonstrated by dropping a little piece of fresh liver tissue into dilute H peroxide solution. In this experiment, pieces of liver tissue will be put into different temperatures of H2O for 5 proceedingss. After that the liver tissues will be placed into separate solutions of H peroxide and the sum of O gas produced in a minute will be measured utilizing a gas syringe.

Hypothesis:

Temperature is a measuring of the grade of heat or coldness of a organic structure or environment. More specifically, it is a step of kinetic energy in a sample of affair. On a molecular degree, temperature is the consequence of gesture of atoms which make up a substance. As the temperature increases, the gesture besides increases. The gesture may be due to external energy applied to the atom of internal energy from the quiver of the atom. As temperature is increased, molecules have increased active energy and reactions between them and the chance that the atoms will clash with each other will besides be greater, this increasing the rate of reaction. In chemical reactions, for every 10A°C rise in temperature, the rate of reaction about doubles. This belongings is known as the temperature coefficient of a chemical reaction. However in a enzyme catalyzed reaction the consequence of temperature is more complex, for proteins change form by heat. There are many factors that can impact the construction of a protein such as temperature and pH. When a protein is exposed to heat, it causes the atoms to vibrate violently, interrupting and upseting bonds within the protein, hence altering the chemical features of the protein. I hypothesize that as the temperature of the H2O bath that the liver tissue is exposed additions ; the sum of O gas liberated will besides increase up. I believe that there will be an optimal temperature for the enzyme and traveling base on balls the optimal degree will do a drastic lessening in enzyme activity ( less O gas will be produced ) . Since catalase is found in about all living things, including worlds, I predict that the optimal temperature for catalase will be

Variables:

Independent Variable – Temperature of H2O bath liver tissue is placed In ( A°C )

Dependant Variable -Volume of O produced in a minute ( ml/min )

Controlled Variable

Concentration of the Hydrogen Peroxide

Volume of Hydrogen Peroxide

Mass of liver tissue

The concentration of H peroxide must be kept changeless because harmonizing to the Collision Theory proposed by Max Trautz and William Lewis in 1916 and 1918, increasing the concentration, increases the opportunities of atoms hitting each other. The volume of H peroxide should besides be kept changeless. Increasing the volume of H peroxide increase the substrate concentration and therefore increasing the rate of reaction. Finally the mass of liver tissue should besides be kept changeless to seek command the sum of enzyme molecules present. Increasing the figure of enzymes agencies there are more active sites present and substrate molecules do non hold to “ line up up ” for entree to an active site. Ultimately increasing enzyme concentration can besides ensue in an addition in rate of reaction therefore the mass of the liver tissue should besides be controlled.

Equipment:

Equipment

Measure

Notes

Digital Stop Watch

1

Thermometer

1

i‚± 0.5A°C

Digital Balance to two denary topographic points

1

i‚± 0.01g

Conic Flask

7

250ml

Beaker

1

500ml ( for H2O bath )

Gas Delivery Tube

1

Gas Syringe

1

i‚±0.5ml

Retort base

1

Clamp

1

Boss

1

Bench Mat

2

Safety Goggless

1

Deionized Water Bottle

1

Package of Ice

1

Used for temperature below 30A°C

Matchs

1

Used to light Bunsen Burner

Chemicals

-Dilute Hydrogen Peroxide

H2O2

1

Concentration ( 2M )

Volume ( 800ml )

Safety Note:

Eye protection should be worn at all times

If liquid gets into oculus, flood the oculus with a soft running pat for 10 proceedingss and seek medical attending

If hydrogen peroxide is spilt in the lab, cover it with mineral absorbent. Dilute with H2O and wash liquid.

Hydrogen peroxide should be stored in a dark brown bottle and attention must be taken when taking the cap as it is possible that force per unit area may hold built up inside it.

Method:

Draw up a suited tabular array or tabular arraies to enter the consequences.

Carefully cut 7 pieces of cow liver tissue utilizing a knife and a cutting mat.

Weigh each piece of liver tissue carefully on the electric balance. Make certain each liver tissue weighs approximately around 0.5 gms.

Place each liver tissue into a separate boiling tubing and add 40ml of deionized H2O to each boiling tubing one time the liver tissue is situated at the underside of the boiling tubing.

Topographic point the warming mat on the tabular array with the tripod on top of the warming mat. Gently place the gauze on the tripod. Once this is done, place the beaker on the tripod and easy heat up the H2O with a Bunsen burner. Put a boiling tubing with a liver tissue sample into the H2O and put a thermometer in the tubing. Heat the beaker until liver sample solution reaches 70A°C. Measure temperature of H2O with a thermometer.

After that, carefully mensurate out 100ml of H peroxide with a mensurating cylinder and reassign the solution to a 250ml conelike flask.

Connect one terminal of the gas bringing tubing to the gas syringe and the other to the conelike flask

Remove the liver tissue from the boiling tubing with a brace of pincers and topographic point it into the conelike flask with the H peroxide.

Quickly cork the conelike flask once the liver tissue is dropped into the solution of H peroxide. Get downing clocking the clip once the liver tissue touches the H peroxide solution.

Stop the halt ticker after 1 minute and record the sum of gas produced. Read off the gas syringe.

When the reading is taken, take the cork and dispose of the H peroxide in the chemical waste container.

Repeat the above stairss until informations points from 10A°C to 70A°C are recorded.. For readings below 30A°C, cool the liver tissue sample with an ice bath.

Diagram:

Consequences:

Table of Consequences

aˆˆ

Volume of Gas Produced in a Minute ( milliliter )

Temperature ( A°C )

Trial 1 i‚±0.5ml

Trial 2 i‚±0.5ml

Trial 3 i‚±0.5ml

Averagei‚±1 milliliter

20

32.0

33.0

35.0

33

30

40.0

36.0

41.0

39

40

45.0

47.0

50.0

47

50

54.0

52.0

54.0

53

60

63.0

60.0

65.0

63

70

43.0

37.0

40.0

40

80

4.0

2.0

4.0

3Table 1.0 – Raw Data

Table 1.1 – Qualitative Observations

Temperature ( A°C )

Observations

20

Effervescene, soft bubbling in solution

30

A Effervescene, soft bubbling in solution

40

A Greater effervescence, more bubbling in solution

50

A Vigorous effervescence and bubbling

60

Violent effervescene, violent release of gas, bubbling in solution

70

Effervescene, soft bubbling in solutionA

80

Bubbling in solutionA

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 H2O bath the liver tissue sample was put it and the sum of O gas liberated from the sample after dropping it in dilute H peroxide in 1 minute. The graph glade shows that as the temperature increases, the sum of gas besides increases up to 60A°C. From 60A°C onwards, the sum of O gas produced decreases drastically and there is a downward incline of the curve.

Discussion:

From the informations obtained, there is an addition of O produced as the temperature of the H2O bath additions. This tendency nevertheless merely applies to the information points from 20-60A°C. At 70A°C nevertheless, there is a important bead in the sum of O gas produced and at 80A°C, the sum of O gas produced is less than 5ml. From the graph, the relationship is clearly represented. Up to about 60A°C the sum of O gas produced additions and ten-degree rise in temperature is accompanied by 6-7ml additions in O gas produced. The sum of O gas produced lessening at high temperatures as shown from 70-80A°C. So as the temperature rises, the sum of enzyme progressively lessenings and the sum of gas produced is less. As a consequence of these two effects of heat on enzyme, there is an evident temperature for an enzyme. Using the graph, the optimal temperature of catalase is about at 60A°C.

The belongingss of a protein greatly depends on its three dimensional form of the molecule. Exposure to heat causes the atoms to vibrate violently and this can do bonds within the protein between different amino acid to interrupt, ensuing in a loss of the proteins biological belongingss. This is known as denaturation of a protein. Heating causes a protein ‘s biological belongingss to alter such as optical rotary motion, form of active site and bonding. The active site of the enzyme is what defines the enzyme. If the active site alterations, the substrate molecules will no longer suit the active site of the enzyme. This is shown in the diagram below.

A protein ‘s stableness 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 degrees of protein construction each playing a important in the stableness of the protein. The primary construction of a protein is the sequence of amino acid residues attached by peptide linkages. Proteins differ in the assortment, figure and the order if their component amino acids. Changes is merely one specific amino acid in the sequence of a protein can change a protein ‘s belongings. The secondary construction of a protein develops when parts of the polypeptide concatenation take up a peculiar form. The most common forms are either gyrating to organize I± spirals or into I? sheets. The third construction of a protein is the typical and precise construction, alone to that specific protein. This is normally the consequence of farther folding and interactions within the molecule. The form is held together for good by four different types of bonds: H adhering between hydroxyl and ketone functional groups, Van der Waals forces between neighbouring atoms, disulphide bonds between two cysteine side ironss and ionic bonds between oppositely charged ions. The stableness of a protein is dependent of the balance of these three constructions. Changing the balance of forces that maintains the native conformation of the protein will take to denaturation.

Decision:

The hypothesis put Forth before the experiment was right. As the temperature of the H2O bath that the liver tissue is exposed additions ; the sum of O gas liberated will besides increase up to a certain degree. That degree would be the optimal temperature of the enzyme and the optimal temperature of catalase deduced from the experiment is about 60A°C. Pass the optimal temperature, the sum of O gas produced drastically decreases as the enzyme is get downing to denature. At 80A°C, the enzyme is about to the full denatured and merely a little sum of O gas is produced. The literature value for the optimal temperature of catalase in homo is about 37A°C which is the organic structure temperature of a typical human being. The liver sample was taken from a cow and both cattles and worlds are able to modulate their ain temperature. From this we can infer that the optimal temperature of catalase for a cow should be approximately similar to a homo ‘s and someplace around 37 -40A°C. The optimal temperature obtained from the experiment is 60A°C, this suggests that there may hold been divergences or fluctuations within the informations.

Restriction to Experiment:

There are several restrictions to our experiment to the experiment. One of the chief restrictions was commanding the temperature of the H2O baths. The temperature began to drop bit by bit one time the furuncle tubings were placed into the beaker. This may hold caused divergence and fluctuations within the information. Another restriction was that the experiment was merely perennial 3 times and there may still be room for anomalousnesss and mistakes.

Evaluation:

Equally long as the temperature of the H2O baths were kept changeless and the sum of gas produced was measured and read right, a good estimate of the optimal temperature of catalase in a cow can be found. However there are several cardinal betterments that can be made. When the liver tissues were cut a batch was wasted and some of it got stuck to the spatula and to the top of the trial tubing. To pack the job in the close hereafter, the liver tissues stuck onto spatula could be washed off utilizing a bantam sum of H2O or shook gently to seek take some of the liver tissue. The method for collected the O gas could besides hold been improved. Some gas was lost due during the clip it took me to infix the spile into the trial tubing. To minimise the sum of gas lost, have a spouse put the spile on for you while you drop the liver tissue into the measurement cylinder. The temperature of the H2O baths started to drop after a few proceedingss and this may hold lead to some little divergence within the information. In future make certain that the temperature of the H2O bath does non alter excessively significantly. A little alteration would be to cover the beakers with a palpebra to forestall air from get awaying. Another job that arose during the experiment was maintaining the temperatures of the H2O baths changeless during trials and between tests. The job was that different temperatures of liver samples were tested and it would n’t be executable to utilize a electronically controlled H2O bath to execute each trial. The consequence was that a Bunsen burner was used but this creates several jobs. It was highly hard to acquire exact temperatures and keep them throughout the experiment. In future, the usage of a warming home base would be more practical so as to forestall drastic alterations in temperatures. To better the experiment it may be better to utilize a solution of catalase instead than a liver as a beginning of catalase. By utilizing a solution, the concentration and volume could be controlled and would be the whole experiment into a just trial. Overall the consequences obtained have helped back up the hypothesis put Forth before the experiment. Finally the experiment could hold been repeated a few more times so that a better norm could be drawn from the informations, ensuing in a more dependable and accurate decision.

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