An investigation to see how different concentrations of aspirin affects hydrogen peroxide breakdown by liver catalase

Introduction:

Aspirin is one of the most widely used medicines. It is very affective in pain relief, and also provides anti-inflammatory action. However, it is known to have side affects. Repeated use may cause gastrointestinal bleeding, and larger doses can cause vomiting, diarrhoea and hallucinations. The key compound in aspirin is salicylic acid. The average dose 0.3-1g, however 10g or more, in a dose, can be fatal. This is a molecular diagram of aspirin:

COOH-C6H4-OCOCH3

H H

OH C C

C C C O

O C C

CH3

H H

Catalase is an enzyme that speeds up the breakdown of hydrogen peroxide into water and oxygen. Enzymes are biological catalysts that are necessary for life. Without these enzymes, life, and the processes involved, would not be supported. Catalase is a large enzyme, containing about five hundred amino acids. It has one of the highest turnover rates of all the enzymes; one molecule of catalase can convert six million molecules of hydrogen peroxide into water and oxygen each minute. This is the reaction that catalase catalysis:

2H2O2 2H2O + O2

Catalase is present in peroxisomes, which are large organelles inside cells. Peroxisomes are involved in the oxidation of fatty acids in animals. Catalase, like all enzymes, is affected by certain factors such as temperature and pH, as well as substrate concentration, salt concentration, and of course inhibitors and activators. Temperature tends to increase the rate of chemical reactions. This is due to the increase in kinetic energy that the substrates have. Heat gives energy to the substrates, making them move faster. The substrates are then more likely to collide, and will have more energy when they collide. This increases the rate of the reaction. That is, of course, up to the point where the enzyme is denatured. It is therefore possible to slow down the breakdown of hydrogen peroxide, to a more measurable rate, by controlling and keeping the temperature relatively low. It is sensible to remember that temperatures above 40-50°C, denatures most enzymes. The pH can change the shape of an enzyme by taking away, or adding, hydrogen ions to its molecules. If pH is too extreme, the enzymes can be denatured, thus reducing enzyme activity. Increasing substrate concentrations, can lead to increases in reaction speeds. This is due to increased probability of the substrate coming into contact with enzymes. Salt concentration can also affect enzyme activity. Every enzyme has an optimum salt concentration at which it works best. Too high or too low concentrations of salt can lead to denaturisation. However, the factor that I will be testing is to do with the presence of inhibitors or activators. These molecules can interact with an enzyme, either causing the enzyme to work better, or to slow down. There are two types of inhibitors; non-competitive and competitive inhibitors. Non-competitive inhibitors work by deforming the shape of the active site, or the whole enzyme, which nevertheless deforms the active site. Competitive inhibitors are molecules which have a similar structure to that of the substrate. If the competitive inhibitor meets an enzyme, it may temporarily block the active site.

Hydrogen peroxide is a by-product of oxidation of fatty acids. Hydrogen peroxide is a potentially dangerous chemical that must be broken down by catalase, to avoid damage to the cell. When hydrogen peroxide is decomposed, heat and oxygen are given out. When working with hydrogen peroxide, care must be taken due to the heat. However, in dilute solutions, the heat is rapidly absorbed, so is not a problem. Hydrogen peroxide is miscible with water, so diluting the hydrogen peroxide with ordinary water is a good way to reduce the dangerous affects of the liquid. There should be adequate ventilation when working with hydrogen peroxide, and there should be easy access to an ample water supply for thorough flushing of accidental spillage on surfaces and on people. Hydrogen peroxide is a non flammable liquid, however the decomposition produces oxygen that is flammable. If fires occur, involving hydrogen peroxide, they are best treated using water.

Reye’s syndrome is a deadly disease that can attack any child or adult. The causes and cure are unknown, but research has proved a link between Reye’s syndrome and aspirin use or the use of the main ingredient in aspirin, salicylic acid. Reye’s syndrome attacks all body organs, with the most damage being inflicted on the brain and liver. There is, therefore, a link between aspirin and liver. I shall construct a test to see if aspirin affects one of the main enzymes in liver, catalase.

Hypothesis:

Increasing the concentration of aspirin will decrease the rate of breakdown of hydrogen peroxide into water and oxygen by liver catalase.

Risk assessment:

There are certain risks in this experiment that I must be constantly aware of. These are outlined below.

I will be constantly aware of any hazards, and will know exactly what to do if something goes wrong. There is a first aid kit in the laboratory and a qualified first aider will be nearby.

Control of variables:

Concentration of aspirin

This is a preview of the whole essay

Risk assessment:

There are certain risks in this experiment that I must be constantly aware of. These are outlined below.

I will be constantly aware of any hazards, and will know exactly what to do if something goes wrong. There is a first aid kit in the laboratory and a qualified first aider will be nearby.

Control of variables:

Concentration of aspirin

I will vary this by changing the concentration of the aspirin solution in which the beads are soaked. I will change the aspirin concentration to see how the rate of hydrogen peroxide breakdown is affected. The average dosage of aspirin tablets taken at any time is from 1-3 tablets. I will therefore use 7 different concentrations. 0, 0.5, 1, 1.5, 2, 2.5, 3 tablets.

Concentration of liver catalase

This will be kept constant. Equal volumes of Ox liver will be used in each test. I cannot measure the catalase volume directly, but catalase volume should not vary much within a small section of the liver. I will find out the volume of Ox liver to use in the final experiment from my pilot experiment.

Concentration of H2O2

This will be kept constant. I will make batch of H2O2 solution, which I will use throughout the experiment. This reduces any errors which might occur in the production of the H2O2 solution. I will do a pilot experiment to find out the optimum H2O2 concentration to use in the main experiment.

Volume of liver

It is difficult to cut liver into similar size portions so I will liquidise the liver in a food processor. I will then use a syringe to create beads of liver and sodium alginate. Once the beads are made, I will sort them into similar sizes. This will hopefully keep the volume of liver roughly the same throughout.

Volume of H2O2

This will be kept constant. I will use a measuring cylinder to measure out 10cm3 of H2O2 solution. 10cm3 is about 2/3 of a test tube, which is roughly what I want. This should be deep enough for me to see when the bead starts to float up, so that I have time to prepare to stop the timing.

Temperature

I will keep the temperature constant by using a water bath for all the tests. I will set the water bath to about 25°c. The actual temperature does not matter, but keeping it the same does. I have chosen this temperature as it will make my experiment easier. There is no risk of burning myself or other people. The heater will have no trouble keeping the water at this temperature as it is slightly above room temperature so little heat will be given off to the surroundings. The breakdown of H2O2 is exothermic. This means heat is given off by the reaction. A water bath will disperse this energy, keeping the temperature constant.

Acidity

I will keep the acidity constant by using a buffer. I will use a buffer of pH 7.4 as this is closest to the acidity of blood. This means I hopefully won’t denature the enzymes.

Bead diameter

I will keep the bead diameter as constant as possible. I will measure the diameter using a ruler, to the nearest half a millimetre. I will use beads of very similar diameters.

Method for pilot experiment:

These are the things that I am trying to find out in the pilot experiment:

Concentration of H2O2 to use
Concentration of liver in beads

Increasing the H2O2 concentration increases rate of reaction. This is due to a higher concentration of substrate, which increases the probability of the substrate meeting the enzyme.

Increasing liver concentration increases rate of reaction. This is due to a higher concentration of enzyme, which increases the probability of an enzyme meeting the substrate.

It is a lot easier to change the concentration of H2O2, than it is to change the concentration of liver. I am aiming to get a time for the reaction that is not too fast, i.e. the reaction happens within a few seconds, as it might be difficult to notice a change in rate of reaction. But I am also aiming to get a reaction that is not too slow, i.e. the experiment does not take a couple of minutes, as I will soon run out of time.

Method:

I have devised a method that will allow me to see the affect that aspirin will have on H2O2 breakdown. I will create beads of liver, which I will then soak in different aspirin concentrations. The aspirin will then (or possibly not) denature the catalase in the liver. I will then take the beads out and drop them into some hydrogen peroxide solution. The catalase will then start to breakdown the hydrogen peroxide into water and oxygen. The oxygen gas created will form bubbles on the surface of the bead, which will hopefully make the bead start to float up to the surface of the solution. I will measure the time it takes for the bubble to reach the surface from when it reaches the bottom of the test tube.

Create the beads:

Weigh out a certain amount of Ox Liver. I will start off with 5g of liver.

Place in a food processor and add water. I will start off with 100cm3 of water.

Process the mixture for 2 minutes until liver is broken down

Filter the mixture into a beaker

Mix together:

1cm3 of liver solution

1cm3 of pH 7.4 buffer. This is the pH of blood. In the human body, the reaction that I am testing occurs where there is a lot of blood. It is therefore safe to say that this pH will not denature any of the substances that I am using.

3cm3 of 4% sodium alginate

Using a syringe, drop small beads of mixture into 0.1 mol dm-3 CaCl2 to harden. The calcium chloride concentration does not matter.

Wash the beads

Select beads of the same size

Put 10cm3 of the highest possible concentration of H2O2 into a test tube.

Drop a bead into the test tube.

Once the bead has reached the bottom of the test tube, time how long it takes for the bead to reach the surface again.

If the bead does not reach the bottom of the test tube or the time taken is very small, dilute the hydrogen peroxide. Record the results. Repeat the process with the new hydrogen peroxide until a reasonable time for the reaction is reached.

Table 1: Results of my pilot experiment

I did not need to change the liver concentration in the beads.

I will use the following concentrations and volumes in the final experiment.

Volume of hydrogen peroxide 10cm3

Concentration of hydrogen peroxide 0.05 mol dm-3

Concentration of ox liver in processor 5g : 100 ml water

Ratio of liver : buffer : alginate 1:1:3

Method for final experiment:

These are the stages in the method:

Make Beads:

Weigh out 5 grams of Ox Liver

Place in a food processor and add 100cm3 of water

Process the mixture for 2 minutes until liver is broken down

Filter the mixture into a beaker

Mix together:

2cm3 of liver solution

2cm3 of pH 7.4 buffer. This is the pH of blood. In the human body, the reaction that I am testing occurs where there is a lot of blood. It is therefore safe to say that this pH will not denature any of the substances that I am using.

6cm3 of 4% sodium alginate

Using a syringe, drop small beads of mixture into 0.1 mol dm-3 CaCl2 to harden

Wash the beads

Select beads of the same size. Larger beads have a smaller surface area : volume ratio. Most of the reactions that take place will occur on the surface of the bead, giving off oxygen, which forms bubbles on the surface. If the bead is large, more oxygen will have to be created to reduce the density of the bead to make it rise. Beads must be similar sizes, to reduce variability in the results.

Make 0.05 mol dm-3 H2O2 solution:

Measure out 5cm3 of 1mol dm-3 H2O2

Add 5cm3 of pH 7.4 buffer

Add 90cm3 of water

Put 10cm3 of 0.05 mol dm-3 H2O2 solution into 7 test tubes.

Making different aspirin concentrations:

Put 10cm3 of water into a test tube

Add soluble aspirin 300mg tablets to solution

Table 2: A table showing the range of aspirin concentrations that I will soak the beads in.

When aspirin is fully broken down, drop 4 beads into the test tube and leave for 5mins

Remove beads from aspirin solution

Timing:

Drop bead into the 10cm3 of 0.05 mol dm-3 H2O2

Record the time from when the bead reaches the bottom of the test tube to when it reaches the top again

Repeat the experiment with different aspirin concentrations

I will then repeat the experiment again a few times. Repeating an experiment enable me to test the control of variables. If I get results the second time, that are similar to the results obtained in the first time, then the method is good as the variables have been fully controlled. However if the repeats give variable results, then some of the variables have not been controlled properly. I will repeat the experiment to test the reliability of my method.

Results:

Table 3: A table showing the times taken for the beads of different aspirin concentrations to rise to the surface of 0.05mol dm-3 H2O2 at pH 7.4.

NB: Data in red are anomalies

Table 4: A table showing the processed results; Mean time, Rate of reaction and Standard deviation of the experiment.

The means and rates were calculated, by excluding any anomalous results.

Table 5: A table showing error boxes boundary calculations, which will be used to draw the graph.

Graphs of results

Analysis

Trends:

From my results, and the graph that I have plotted, I can see that aspirin decreases the rate of hydrogen peroxide breakdown by liver catalase into water and oxygen. The catalase reduces the activation energy required to break the bonds in the hydrogen peroxide molecules. As the concentration of aspirin increases, the rate of breakdown reduces. Between 0 and 15 gdm-3 there is a large reduction in the rate of breakdown. After this, from 15 to 90 gdm-3, there is less of a reduction, but a reduction is definitely still present. Despite frequent overlaps between error boxes of similar concentrations, there is still an overall reduction. Between 0 and 15 gdm-3 there is a decrease in the rate from 0.0427 to 0.0314, this is a reduction of about 25%. However, between 75 and 90 gdm-3 there is only a decrease from 0.0239 to 0.0211, which is a reduction of about 12%. The hydrogen peroxide molecules come into contact with the enzyme. The active site of the enzyme is the area where the hydrogen peroxide molecule is broken down. Active sites have a complimentary shape to the substrate molecule. The catalase provides a surface for the substrate, hydrogen peroxide, to attach to, where it then breaks down. The bonds are broken, and then reformed to form different molecules. The aspirin molecules bind with the catalase enzyme. This stops the catalase from doing its job, thus reducing hydrogen peroxide breakdown rate. However, I do not know whether this inhibition is competitive or non-competitive. Thinking about this logically, hydrogen peroxide molecules contain only four atoms, two hydrogen, and two oxygen. Aspirin molecules are much larger; they consist of 21 atoms. I can tentatively say that aspirin is too large to fit into the active site of catalase, and is thus a non-competitive inhibitor. Further tests would need to be carried out to prove this. If the inhibition is non-competitive, the aspirin binds to the catalase molecules, changing it’s tertiary structure. This stretches the enzyme, which results in the active site changing shape. The active site no longer has a complimentary shape to the hydrogen peroxide, so is denatured. There is a reduction in formation of an enzyme substrate complex. The main ingredient in aspirin is salicylic acid. From its name, I can tell that salicylic acid has a pH below 7. I controlled the pH of my experiment at all times to pH 7.4. I chose this pH as it is the pH of blood, so the effects shown are not due to inhibition by reduced pH but by some other type of inhibition.

Anomalous Results:

I have identified some anomalous results in my experiment. The anomalous results are the ones that don’t fit into the pattern and trends. Anomalous results tend to affect means and standard deviation. They are highlighted in red in Table 3. The fifth time for 0 gdm-3 aspirin concentration is an anomaly. The bead reached the bottom of the test tube, but then started to rise almost immediately. I had a look at the bead and discovered that there were a few bubbles in the bead. The bubbles in the bead were due to air captured in the syringe in the production of the beads. The bubbles reduced the density of the bead, thus causing the bead to rise when less O2 had been produced. I ignored this result because otherwise it would have produces a slower mean rate.

There were two anomalies in the test for 60 gdm-3 aspirin concentration. The first anomaly gave a much faster rate. This was unexpected. I noticed that the time taken was very similar to the time taken for 0 gdm-3 aspirin concentration. I immediately realised that I had accidentally put a bead into the test tube that had not been left in the aspirin solution. I ignored this result.

The other anomaly in the test for 60 gdm-3 aspirin concentration gave a much slower rate of reaction. I can not explain why this result was different, but it is likely that a variable was not properly controlled. This was most probably bead size.

Evaluation

Reliability:

I calculated the standard deviations of my data. This enables me to analyse the variability of my data. The standard deviations are fairly low, never exceeding 3. This shows that there was low variability in the repeats, thus showing that the method, and techniques used were fairly reliable. There was some variability, which I think is due to the difference in bead size more than anything else. Bigger beads have a greater mass, and are thus heavier. This means more oxygen has to be created to reduce the density of the bead, and make it rise. The smaller the bead, the faster it will rise. The variability in my results means that my method can be improved. I have described some possible ways in which I could improve my method in the improvements section below. I have chosen to remove any anomalous results from calculations. I have compared the means, rates, and standard deviations of all the results, with the calculations excluding anomalies. The anomalies would have affected the results, had I not ignored them when calculating the means.

I believe the results that I have obtained enable me to tentatively accept my original hypothesis; increasing the concentration of aspirin will decrease the rate of hydrogen peroxide breakdown into water and oxygen by liver catalase. However, I cannot produce a quantitative conclusion based on my results. To do this, I would need to take a lot more results.

Limitations of the techniques:

There are lots of limitations in my technique. I do not think that the varied quantities of aspirin were over a wide enough range. It might be that aspirin starts to speed up the rate of reaction at very high concentrations. Another limitation was the use of beads. This is not a very accurate way to calculate the rate of reaction. The catalase within the beads would have already started reacting with the hydrogen peroxide before the bead reached the bottom. The beads were very probably not all the same size. The beads were not formed from certain volumes or masses; they were just created and compared for similar sizes using a ruler. This is a subjective method. I used beads that had a diameter of about 4mm in this experiment, as this was the size that I had the most of. When measuring my beads, I could only measure them to the nearest 0.5mm. This gives an error of = 12.5%, which is fairly large. If a gas syringe were to have been used, it would have been possible to measure all the oxygen given off within a certain time limit. The accuracy of the syringes was also a problem. Syringes are accurate to about 0.1cm3. In my method, I had to measure out 5cm3 of liquids. The error is reasonably small, = 2%, but it is still a source for variability in my results. Another limitation was that I couldn’t control the temperature well enough. I could not use a water bath as one was not available. The use of a water bath would provide less fluctuation in temperature, and thus less variable results. All this would lead to more accurate data that is less limiting in drawing a conclusion.

Improvements:

There are many improvements I would make to this experiment in order to make a correct conclusion. I would not change what I was investigating, as there are still some unanswered questions in my current experiment. Probably the least well controlled variable is the size of the beads, i.e. the volume of liver. I would measure the beads more accurately with vernier callipers. Vernier callipers are accurate to 0.02mm. I would not need this degree of accuracy for my beads as it is very unlikely that I can make beads that only have a difference of 0.02mm in their diameters. I would measure the beads to ±0.5mm, and then group them into very similar sizes. However, I would need to be careful that I do not crush or squash the beads when measuring them. Measuring the beads to a greater degree of accuracy would mean less variability in the results, and would enable me to write a much more accurate conclusion.

Another variable that was not very well controlled was the temperature. I did not have access to a water bath, so was unable to keep the temperature exactly the same. I thought this would not matter as I did not feel that changes in the temperature, would affect the rate of hydrogen peroxide breakdown. This might be true, but next time, I would control the temperature better. The reaction, 2H2O2 2H2O + O2 is exothermic. This means heat is given out by the reaction, which inevitably increases the temperature if the heat is not dispersed rapidly enough. A water bath would do this. For my first set of repeats, I put the beads into test tubes that had already been used for a test. I did not think that this would matter, but as the reaction is exothermic, the temperature might have been greater for the repeats. However, this is not reflected in my results. Increasing temperature tends to increase the rate of reaction, but my results do not show an increase in rate for the later repeats. If I were to do this experiment again, I would use water baths.

I think the variability in my results is mainly to do with the difference in bead size. But I would still control these other variables better as well.

Conclusion:

On the basis of this one experiment I think I have enough evidence to tentatively accept the hypothesis that increasing the concentration of aspirin will decrease the rate of breakdown of hydrogen peroxide into water and oxygen by liver catalase.

However, to be sure I would need to repeat the experiment many times with the improvements to the method.

Affect of aspirin on catalase /