Effect of enzyme concentration on initial rate of reaction

The objective of this experiment is to determine how differentiating the concentration of the enzyme catalase affects the initial rate of reaction, in the decomposition of Hydrogen Peroxide (H2O2)

The equation of this reaction is:

Hydrogen Peroxide(aq) → Water(l) + Oxygen(g)

2H2O2(aq) + catalase(aq) → 2H2O(l) + O2(g)

The source of the enzyme Catalase in this experiment will be yeast, specifically the Saccharomyces Cerewisea strain, as it is easy to obtain and very safe with no harmful effects to the environment.

The technique used to obtain a set of results will be the downward displacement method, where evolved oxygen product from the reaction is passed through a delivery tube and into a water filled burette, pushing the water out of the burette, leaving obtainable results of Oxygen collected. These results will be taken every 5 seconds for two minutes and will give the rate of reaction.

My Equipment will be:

Burette
Side-arm Conical Flask
Bung
Tubing
Yeast
Hydrogen peroxide
Water Bath
Water/Margarine Tub
Distilled water/pH7 buffer
Stopwatch

I chose to have a burette to collect the evolved Oxygen, as it has a great degree of accuracy. The gas can be measured to 0.05ml or 0.5cm3.

I chose to have a side arm conical flask to provide a source for the evolved oxygen to travel through easily. Yeast is the chosen source of the enzyme as it is easier to measure than potato slices, as it will be in liquid form and can be measured with a graduated pipette. Also, there will be an even distribution of enzyme and a constant surface area to work in.

Diagram of the setup

Variables/Background Info

Temperature – As temperature rises, the rate of reaction also increases. This happens because the molecules have more kinetic energy (according to Kinetic Theory), so they collide more often, and more of these collisions result in the substrate binding to the enzymes active site. This means that there is more chance of a reaction occurring so the rate of the reaction increases. However if you increase the temperature too much the rate of reaction will decrease. This happens because the active site of the enzyme begins to distort, just like with pH, after a certain temperature, depending on the specific enzyme. The hydrogen bonds, and possibly the disulphide bridges present in the enzyme can break, so the active site changes. This is because when the aforementioned bonds are broken, the structure of the whole enzyme changes, and therefore no enzyme substrate-complexes are able to form. Thus, no product is formed. We say that the enzyme has been denatured, this happens at around 40oC for most enzymes, especially in animals.

This variable will be investigated further to obtain an optimum temperature that the source of Catalase works at, then this optimum temperature will be kept at a constant.

Pressure – increasing the pressure can also increase the rate of reaction, this is because the molecules are pushed closer together, and therefore collisions are likely and will occur more often between the molecules, like when you increase the concentration. Hence there is a higher chance of a reaction happening. The pressure in this experiment will be the same at 100kPa (100,000 pascals)

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Inhibition – inhibitors either compete against the substrate for the active site of the enzyme, or bind themselves to the enzyme, altering the shape of the enzymes active site, preventing the substrate from binding to the active site and consequently stopping it from functioning properly. These are competitive and non-competitive inhibitors respectively. Inhibitors should not be a problem in this experiment as they will not be present.

pH – Any change in the pH of the Solution alters the ionic and hydrogen bonding in an enzyme, and thus alters the overall shape. Similarly to temperature, each different enzyme has a different optimum temperature that it works quickest at. This is when the active site best fits the substrate. If the pH is too extreme for the enzyme, it is denatured, again similarly to temperature. In this experiment, the pH will be pH7, as suited to its natural environment, in potato and the liver.

Enzyme Concentration – an increase in enzyme concentration should lead to an increase in rate of reaction correspondingly, provided there is an excess of substrate. However, when the substrate is a limiting factor, i.e. when it is in short supply, an increase in enzyme concentration will have no effect when the reaction has slowed, as there will be no substrate to bind to these active sites. This will be the only independent variable, after the preliminary experiment has taken place

Substrate Concentration – an increase in substrate concentration should lead to an increase in the rate of the reaction accordingly, providing the substrate is in excess. However, when the enzyme’s active site is in short supply, an increase in substrate will have no effect. The substrate concentration will be constant throughout the experiment to ensure a fair test. This may be adjusted in the preliminary stages to gain the most convenient concentration

The above factors will be kept at constant where necessary to ensure all the results are fair, and therefore get overall more accurate results.

Preliminary work:

To be certain of the optimum temperature that the enzyme catalase works at, I will do 3 experiments at 5 different controlled temperatures, to be certain of a fair test and the true optimum temperatures. These temperatures are 4˚C, 25˚C, (room temp),30˚C, 40˚C, and 50˚C. The most sensible educated prediction as to which will be the optimum temperature would be between 30˚C and 40˚C, as these are the temperatures which yeast occur naturally. The amount of oxygen formed will be recorded every 5 seconds for two minutes and record the results for each temperature, 3 times for each, and the optimum temperature can be seen in which to carry out the final experiment.

A graph of the below shape is expected:

The concentration of the Hydrogen peroxide that will be used must be determined, because if it is too high, the products will be formed too fast to record. The yeast Solution’s composition will be:

1g dry yeast in 50ml of distilled water (to eliminate impurities), and 0.5g of sugar.

Prelim Method:

Set up the apparatus like in the diagram above
Place 5ml Yeast containing Catalase and 10ml hydrogen peroxide in water bath (in different vessels), which will be at specific temperatures.
Remove when at required Temperature.
When yeast and Hydrogen Peroxide at required temperatures, mix the hydrogen peroxide (20vols/10ml) quickly into the side-arm conical flask, and then the 5ml yeast into the same flask, and quickly bung the top.
Measure the oxygen that has evolved every 5 seconds for two minutes, as to gain a value for the initial reaction.
If reaction too fast, half the concentration of hydrogen peroxide (to 10, then 5vols).
Repeat this for the 5 specific temperatures.

Precautions:

It must be assured that all equipment is washed between experiments where variables are changed, such as the conical flask, graduated pipette etc. also, making sure that they are dried thoroughly is important as any excess water could increase the dilution of the reactants and affect the results.

It is also imperative that the stopwatch is started exactly at the same time in each experiment; as soon as the bung is put in.

There are a couple of minor safety issues that must be addressed to assure 100% safety. Hydrogen peroxide can be corrosive, and can act as an irritant to the skin, eyes and clothing if spilled and not washed off thoroughly.

All enzymes are potential allergies and can irritate membranes found in the eyes nose, or any other cavity. They can also cause asthmatic attacks. Spills of any substance should be washed immediately to obtain the safe environment. A lab coat and goggles should be worn to ensure these safety conditions, however inexplicit they may be.

Hypothesis:

Due to factors of natural occurrence of yeast, which include in the liver and potatoes, the suggested optimum temperature will be around 40˚C. This can also be explained through the baker’s method of warming the yeast before cooling it as not to denature the yeast. This indicates the 40˚C mark may well be the optimum temperature. It may also be expected that a lower concentration of hydrogen peroxide may be needed for accurate results to be taken.

The results of this preliminary work will be taken and used in the final experiment. They will be analysed and the temperature which best suits yeast will be used. The independent variable in the final experiment will be enzyme concentration. The graph will be similar to the one of temperature against rate of reaction, in that the initial rate will be high and will slow down just after optimum is reached. However, after this point, instead of stopping completely as in temperature, the graph should plateaux. This will be the point when all the enzymes active sites are saturated.

Therefore, by changing the concentration of the enzyme, it will be possible to see any changes in the rate of reaction. The graph will be analysed and a rate of reaction worked out for each different concentration. 3 tests of each concentration will be taken out to ensure accuracy and fairness. It is likely that as the concentration is increased, the rare of reaction will increase proportionally. This is due to the fact that when enzyme concentration is doubled for example, there will be double the active sites present. So, if the substrate is in excess, theoretically, the rate of reaction should double. This should be indicated by the amount of O2 produced every 5 seconds. the dependant variables in the preliminary experiment will be H2O2 concentration and temperature.

The first round of Preliminary experiments took place with 20vol concentration of hydrogen peroxide at room temperature. This was the chosen temperature to determine the required concentration of Hydrogen Peroxide as was convenient to prepare.

No results were taken due to the rapid nature of the breakdown of hydrogen peroxide; the reaction had finished within 6 seconds, so 10vols was used.

The results were:

Temp: 25˚C

This too was slightly too rapid to record accurate results, so 5vols will be the next tested concentration of the hydrogen peroxide.

The results were then taken at different temperatures with 5vol concentration of Hydrogen Peroxide. The results are below:

Temp: 25˚C

This experiment gave recordable, and fast results, so the concentration of hydrogen peroxide to be used will be 5vols. The 25˚C temparutre will also be used as one of the preliminary experiments to find the optimum temperature.

The next temperature to be tested was 4˚C

The results are below:

This experiment seems to give good results. However, the rate of reaction seems to increase slightly as time goes on. This may be due to the pressure in the delivery tube only building, and this would explain the bigger than average bubbles. Also, the temperature may have been increasing very slightly due to the room temperature. Overall, this temperature is not the optimum as 25˚C was faster.

The next temperature to be tested was 30˚C

The results are below:

These results were similar to the 25˚C experiment, so the graph will show the experiment which has a higher initial rate of reaction.

The next temperature to be tested was 40˚C

The results are below:

This also gave relatively similar to the above experiments but seems a little bit slower. However, it cannot be ruled out until the graph is completed.

The last temperature to be tested was 50˚C

The results are below:

Once again, these resultsare similar to the above, so will again be determined via the graph.

Method final experiment:

Set up the apparatus like in the diagram drawn
Place 5ml Yeast containing Catalase and 10ml hydrogen peroxide in water bath (in different vessels), which will be at the specific optimum temperature.
Mix the hydrogen peroxide (10ml/5vols) quickly into the flask containing yeast (5ml), when at required temperature, and bung the top.
Measure the oxygen that has evolved every 5 seconds for two minutes, after the first bubble has evolved as to gain a value for the initial reaction.
Repeat this 3 times for the specified enzyme concentrations, and work out the rate of reaction for each, and then an average for each concentration.

Teacher Reviews

Here's what a teacher thought of this essay

Stevie Fleming

29th of May 2013

*** Plan and initial experiments only. This mark is awarded only for the planning section. The inclusion of many different trial experiments means that this experiment has been thoroughly planned experimentally to find the optimum values for the temperature and concentration of Hydrogen Peroxide. The hypothesis is testable and is explained using relevant biological background material. The key variables are considered well. To improve: The inclusion of more relevant background material to inform the plan would be helpful. The material from textbooks or websites should be referenced at the end. The risk assessment is not adequate to be awarded marks under the newest marking schemes and needs to considered much more carefully. The writer needs to ensure that all graphs and table headings are fully labeled with a heading and unit. This should also include sketch graphs used to illustrate a point.

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