The activity of Enzyme Concentration on Hydrogen Peroxide.

Introduction

In this investigation, I will be investigating the activity of the enzyme Catalase on the substrate hydrogen peroxide (H2O2).

Scientific Background

Enzymes are biological catalysts that speed up the thousands of metabolic reactions that occur in living cells. They are usually large proteins made up of hundreds of amino acids, and often contain a non-proteinaceous group, known as the prosthetic group. This is what is important in the actual catalysis.

In an enzyme-catalyzed reaction, the substance to be acted upon, the substrate, binds to the active site of the enzyme. The substrate is held to the enzyme in an enzyme-substrate complex by hydrophobic bonds, hydrogen bonds, and ionic bonds.

The enzyme then converts the substrate to the reaction products in a process requiring several steps, and often involves covalent bonds. Finally, the products are released. By allowing this to occur, the enzyme reduces the activation energy required in any chemical reaction. All chemical reactions require energy for them to occur, because there is an energetic barrier that reactants must overcome for them to be converted into the products. Once enough energy is supplied, a position called the transition state takes place, and the reactants can then form the products. This energy is called the activation energy. Enzymes reduce this activation barrier.

Each enzyme is specific for a certain reaction due to its unique amino acid sequence, which causes it to have a unique three-dimensional structure. The active site also has a specific shape, therefore only allowing few of the many compounds present to interact with it. Any substance that blocks or changes the shape of the active site interferes with the activity and efficiency of the enzyme. Catalase, for example, acts only on hydrogen peroxide, and is ineffective on any other natural substrate. The induced fit hypothesis is the recently put forward theory explaining how enzymes work. It shows that the active site may not necessarily be exactly the right shape to begin with. It is believed that when the substrate combines with the enzyme it causes a small change to occur in the shape of the enzyme molecule, thereby enabling the substrate to fit more snugly into the active site. This has replaced the idea originally put forward called the lock-and-key hypothesis, which says that the active site is permanent and does not change shape.

The enzyme Catalase accelerates the breakdown of hydrogen peroxide (a common end product of oxidative metabolism) into water and oxygen. Catalase in one of the fastest enzymes. It has a turnover number of approximately six million, increasing the speed of the reaction by 1014 compared with what it would be in the absence of the enzyme. The reaction process is as follows:

2H202 2H20 + O2

This Catalase-mediated reaction is extremely important in the cell because it prevents the accumulation of hydrogen peroxide, a strong oxidizing agent that tends to disrupt the delicate balance of cell chemistry.

Catalase is found in the peroxosomes (an organelle) of animal and plant cells, and is especially abundant in plant storage organs such as potato tubers and fruits. As with all enzymes, catalase is a protein, meaning that it is synthesized within the cell from building blocks called amino acids. In addition to the amino acids that make up the protein, catalase carries a heme group. In the middle of the heme group, there is an iron atom. The catalase enzyme uses this iron atom to help it break the bonds in the hydrogen peroxide, shifting the atoms around to release two molecules of water and a molecule of oxygen gas.

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Concentration is the amount of a given substance in a set volume. As the concentration of the enzyme increases, there will be more collisions and hydrogen peroxide particles will decompose, producing more oxygen gas. If the concentration of the enzyme is doubled, the number of particles will double in the same area. According to the Kinetic Theory, everything is made of tiny particles that are constantly in motion. In order for a chemical reaction to take place, the reacting species must collide with one another. However, not all collisions may be successful. If they have not got enough energy, the reacting species may just bounce of one another. An enzyme is added to help the reactant come together to make the reaction happen at a lower energy state, and in doing so lowering the activation energy. Therefore, if the concentration of the enzyme is doubled, the number of successful reactions should also double – there will be twice as many particles decomposing at any given time.

References:

www. Madsci.org
Biology 1 – pg 42-47
Chemistry exercise book

Apparatus

Potato – This is where the Catalase was obtained from.
20% Hydrogen Peroxide – 40ml of H2O2 will be used in each experiment.
50ml Measuring cylinder – To give an accurate measure of the H2O2, this will be used in the experiment.
Cork Borer – This will be used to cut out a cylinder of potato, uniform in width and diameter.
Vernier calpier – This will be used to measure out accurately, the amount of potato to be used.
Stop Clock – An accurate account of the initial rate of reaction can be made using this, when the reaction is on.
White tile – This will be used when cutting the potato cylinder into discs.
Razor Blade – This will be used to cut the potato into discs.
Delivery funnel – This will be used to add the H2O2 to the potato. A valve will be used to close the whole in the funnel to stop gas escaping.
Conical Flask – Will be used to hold the contents of the reaction.
Electrical Water Bath – Used to keep a constant environment at 35ºC, in terms of temperature, for the reaction to occur in.
Retort stand, boss & clamp – used to hold the conical flask in the water bath, so that the temperature was constant. This will also be used to hold the gas syringe in place.
100ml Gas Syringe – This will be used to measure the amount of oxygen given off by the experiment.
Water – This will be used to clean equipment and stop contamination.
Beaker – Will be used to hold H2O2 as a stock container.

Method

Once the water bath is filled with water, the temperature is monitored. When the temperature reaches 35ºC, the water bath maintains the temperature.

To prepare the potato discs, first a potato cylinder must be removed from the potato. A cork borer is driven through the potato, giving a cylinder. The cylinder is then measured using a Vernier Caliper. Depending on how many discs necessary, the process is repeated until a suitable amount of cylinders are available. The skin is removed using a razor on each end. The remaining potato is cut into discs 1mm thick on a white tile.

The conical flask is set up so that it is partially immersed in the water bath. It is held in place using a retort stand. The rubber bung connected to the delivery funnel is removed and the potato discs are emptied into the flask. The bung is then replaced.

The discs are then left in the conical flask to equilibrate for 5 minutes. This will allow the enzymes, within the potato, to be working at the temperature of the water bath.

A beaker containing H2O2 will be used to measure out 40ml of the substrate into a measuring cylinder. The measuring cylinder is then emptied into the delivery funnel, giving a more accurate amount of substrate used. The tap will have been closed so that no H2O2 will have dripped into the potato discs.

The tap is opened and, simultaneously the clock is started. When all the H2O2 is emptied the tap will be closed, to stop any gas from escaping. The gas then flows through to the gas syringe, which will take the reading of gas evolved. Measurements of the gas evolved are taken, especially at the start to give a more accurate interpretation of the initial reaction, as at that point the substrate in excess, so this gives the most accurate estimation of what the rate of the decomposition is, when the enzyme is involved.

Readings are taken at 5secs, 10s, 15s, 20s, 30s, 40s, 50s, and 60s.

Once the results have been noted the conical flask is removed, and washed using water. Though the enzymes are reusable, new discs of potato are used in their place. This time, maybe a differing amount of discs is used, giving a more varied enzyme concentration.

This method is repeated for until all results are recorded. For each concentration of enzyme, 3 different readings are taken and an average of the results is taken, if they are consistent.

Safety Precautions

The following safety precautions will be maintained throughout the experiment to maintain a safe working environment.

H2O2 is corrosive, and at 20% is definitely an irritant.
Goggles, lab coat and gloves must be worn at all times.
Care must be taken when using sharp implements.
Any broken glass must be cleaned up immediately.

Variables

The initial rate of a reaction is usually at the start of a reaction, and is the fastest part. It will give the most accurate indication of enzyme activity and rate as at the start, there is an excess of substrate, as a reaction goes on the substrate becomes the product. If rate is measured later on in the reaction, a true indication of activity would not be given of, due to the fact that there is not sufficient substrate to allow to enzyme to work at its optimum rate. This can act as a limiting factor.

However, the rate of a reaction can be affected by many different variables. To make the precision and reliability of the results to a maximum, these factors will be tried to be controlled, so that the only influence on the results is by the factor being tested.

Below are the factors:

Temperature. Due to the fact that enzymes are thermo-labile. This causes it to be affected by differing temperatures. This is due to Kinetic Theory and the basic principal that when more energy is input; the output will display a consequent effect. In this case, an increase in reaction rate. However, after a certain temperature, the enzyme becomes disfigured (denatured), and no longer is specific to a substrate. This inhibits its job, rendering it useless. During the experiment this factor will be kept constant by regulating the temperature of the water bath.

Concentration of substrate. Substrate concentration affects the rate of a reaction. It can be explained using the Kinetic Theory. If there are increasing molecules, the chance of a collision increases, and the chance of a successful collision also increases. However, when excess substrate is added, a maximum rate can be reached. This is due to enzyme concentration being a limiting factor because there are no more enzymes, with vacant active sites to create more products. This will be kept constant by reacting the potato with 40ml solutions of 20% H2O2.

pH. This affects the rate of reaction because certain amino acid sequences interact with solutions, creating a Zwitter Ion at different pHs or Iso-electric Points. This property of amino acids can affect the way an enzyme active site works. The pH will be kept constant using a pH buffer.

Concentration of Enzyme. Enzymes catabolise reactions. Therefore, if the numbers of enzymes increase, surely the rate does too. This is due one again to Kinetic Theory and collisions. However, when there is a limited amount of substrate, it can act as a limiting factor, and may not give a true indication of the rate of reaction. This is why it is best to test the reaction for its initial rate or use excess substrate.

I have chosen to vary the concentration of the enzyme. To do this I will have to vary the surface area of the potato. Uniform sized discs will be used for the investigation. This is because the effective area for the substrate to react with the enzyme is increased. The rate will be indicated by measuring the amount of oxygen given out in a given time.

Predictions

From my relative scientific background and my preliminary study, the following predictions have been made:

As the concentration of the enzyme increases, the initial rate of reaction will also increase. This is because the enzymes have free active sites and there is an excess of substrate. Based on the Kinetic Theory, rate should increase because of the increased likelihood of there being a free enzyme and the chance of a collision increases due to a large amount of particles. The initial rate of reaction should have a graph similar to the predicted one below.

In accordance with the above prediction, if the concentration of catalase is doubled, rate will also double. This is because if there are twice as many enzymes, with an excess amount of H2O2 particles, there will be double the amount of active sites available for the reaction to take place. In theory, this would give a double rate of initial reaction.

This being said, it can also be predicted that there will come a point where the system becomes saturated. This is due to all the active sites being filled and there being H2O2 particles waiting to be decomposed. This is known as the enzyme being limiting factor, as there are not enough to increase the rate, further than that which it has reached.

However, as enzyme concentration is being varied, the enzyme concentration can be increased to meet this need of excess substrate. The same theory can be applied to when there are more enzymes than necessary. This would also cause there to be a maximum rate. In this case substrate is the limiting factor. Therefore, the following predictive graph can be drawn:

Preliminary investigation

Preliminary work was carried out to give a firm backing to base my predictions on and to give an understanding of the most accurate procedure.

Apparatus:

Potato
Stop Clock
50ml glass measuring cylinder
White tile
Safety goggles, lab coat and gloves
H2O2
Beaker
Vernier Caliper
Delivery funnel
Razor blaze
Retort stand, boss & clamp
Water bath
Conical flask
Beehive

Method

The method used was found to be efficient enough to be used in the experiment.

The potatoes however were not cut into discs. They were kept as cylinders.

The length of potato cylinders was varied.

Instead of using a gas syringe a measuring cylinder on a beehive was used. This was found to be the only other inconvenience with the experiment. The displacement of water was used to indicate amount of oxygen given off.

For the preliminary investigation readings were taken at 10s, 20s and 40s

Problems encountered with the method were as follows:

Using the length of potato cylinders was not providing enough exposure of the enzyme to the H2O2; as a result the amount of oxygen given off was not sufficient. This is why discs are going to be used because they each have the equal amount of exposure of any cylinder, no matter what length.
The measuring cylinder on a beehive was just impractical. It was not stable and the displacement was hard to read off accurately. An alternative was to use a gas syringe. This will be used in the experiment because of the practicality.

Results:

The following results were obtained:

Volume of O2 produced/ml

Conclusion

From the results table it can be seen that the initial rates do increase with the increase in length. However, eventually all of the cylinders gave off between 8-9ml at the end.

Though the amount of O2 being produced increases there should be a decreasing rate. However, this cannot be seen from the graph above for “3cm”. This may be due to the cylinder’s lack of exposure to the H2O2, and a substantial result could not have been achieved. The 5cm cylinder does show the predicted trend of an increasing rate, then a constant rate. This gives further support for the prediction. The 7cm also portrays the predicted trend. Though it hasn’t reached its constant rate, the rate is slowing down.

The graph also supports the prediction made that because there is the same exposure of enzymes, no matter what length of cylinder. This is because there is not vast difference between the results. With an exposed surface maybe more dramatic and substantial results will be achieved.

Overall the preliminary investigation was an effective way of supporting predictions made and also trying and improving the method.