The effect of aspirin on the action of bovine liver catalase

Aspirin is a drug commonly used for pain relief and the prevention of heart attacks and strokes. It is readily available over-the-counter and is relatively cheap. The active ingredient in aspirin is acetylsalicylate this compound acts as an antipyretic, anti-inflammatory, anticoagulant and analgesic; this means that is relives pain, fevers, inflammation and prevents blood clots. Aspirin was first discovered around the year 200 B.C. when a Greek called Hippocrates used willow bark (rich in salicin) to relieve pain and fevers. It has since then been refined and compounded to form the modern aspirin.

Aspirin works as an enzyme inhibitor, irreversibly binding to prostaglandin synthase, therefore preventing the production of the hormone prostaglandin. The aspirin is believed to bind covalently to the active site of the enzyme prostaglandin synthase, blocking the substrate from entering the active site although the exact action is not fully understood. The cell must produce more of the enzyme to replace the inhibited enzyme.

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Prostaglandin is released as a response to cell damage (eg. an injury) and encourages inflammation, increase in body temperature (fever), and the dilation of blood vessels. Under normal conditions prostaglandin also controls blood clotting, among other functions, hence aspirin’s anticoagulant properties.

Recent research suggests that aspirin can also reduce the risk of prostrate cancer, breast cancer, leukaemia, colorectal cancer and hogkins disease. In particular the research into the effect of aspirin on the risk of colorectal cancer (cancer of the colon), which involved 27,077 female nurses, has shown that the greater and more regular the intake of aspirin the lower the risk of colorectal cancer. So some people may be beginning to take greater, more regular doses of aspirin to try and prevent the risk of cancer.

However there are some risks involved with taking aspirin, symptoms can include: vomiting, stomach pain, loss of hearing, difficulty breathing, dizziness, mental confusion and drowsiness. This is because aspirin also has effects on other bodily functions including:

o Stimulation of the respiratory centre

o Inhibition of Krebs cycle (respiration)

o Inhibition of amino acid metabolism

Aspirin affects all tissues (as it affects metabolism), especially the stomach where it reaches high concentrations, but also in particular the liver, kidneys and lungs. Heavy drinkers who take aspirin have been found to have an increased chance of liver disease. Another more rare, but more serious illness, Reye’s syndrome, has also been associated with aspirin. Reye’s syndrome causes an abnormal build up of fat around all organs particularly the liver, and causes a severe increase of pressure on the brain. If not treated the patient will often die within a few days.

Catalase is an important enzyme involved in many chemical reactions within the body. Its main use is to break down hydrogen peroxide, a dangerous by-product of metabolic reactions including the oxidation of fatty acids, oxidation of amino acids, purine oxidation and production of DNA. Hydrogen peroxide is toxic and will cause damage to the cell if not broken down after these reactions. When hydrogen peroxide is broken down by catalase it forms oxygen and water, both of which are harmless.

However this is not the only use of catalase, it is also able to break down other toxins including phenols, formic acid, formaldehyde and alcohols using hydrogen peroxide.

Catalase is also one of the fastest acting enzymes, each molecule is capable of breaking down 5 million molecules of H2O2 per second.

Catalase is mostly found in organelles called peroxisomes, although there is also some flowing freely through the blood. Peroxisomes are small organelles similar in structure to lysosomes; inside they contain enzymes involved in the metabolism of amino acids, fatty acids, alcohol and purines and the synthesis of cholesterol and bile acids. Although peroxisomes are found in most cells they are particularly concentrated in liver and kidney cells.

Hypothesis

Aspirin will inhibit the action of bovine liver catalase. This means that increasing the concentration of aspirin will decrease the rate of the catalase-aided reaction which breaks down hydrogen peroxide into water and oxygen.

Control of variables

pH- All enzymes have an optimal pH, any solution that is either more acidic or alkaline than this will cause some of the enzyme to denature. This means that pH must be kept constant and at a value suitable for the enzyme used, the optimal pH of bovine liver catalase is 7.0. The pH will be kept constant using a pH 7 buffer.

Temperature- Enzymes also have an optimal temperature, for mammalian enzymes this is usually around 38 �C, temperatures above this will cause the enzyme to denature. Temperatures lower than the optimal temperature will cause the reaction to be slower, this is because at a low temperature particles vibrate less, so collisions between the enzyme and substrate are less likely to occur. However, because catalase is a very fast acting enzyme it can be used at a lower temperature and still have a suitable rate of reaction. The temperature must still be kept constant so that the rate of reaction is not affected during the experiment. To achieve this the experiment will be conducted in a water bath at a temperature of 30 �C, above room temperature, but below the optimal temperature. All solutions to be used in the experiment will be kept in the water bath for at least 3 minuets before use to ensure that their temperatures are always the same. The breakdown of hydrogen peroxide is an exothermic reaction, hopefully the water bath will disperse the heat created by this reaction.

Concentration of aspirin- This is a varied for different results, but will need to be kept constant for each repeat of the same concentration, and must be accurate. Each concentration will be made by mixing 0, 0.5, 1, 1.5 or 2 aspirin tablets with 30 ml of distilled water. The water will be measured using a syringe, which will give accuracy to the nearest 1%.

Concentration of hydrogen peroxide- The hydrogen peroxide solution will be diluted carefully using 1 mol dm�� hydrogen peroxide solution and distilled water, to the concentration determined in the pilot experiment. The measurements will be made using syringes, which will deliver accurate volumes of the solutions. All of the repeats will be made using hydrogen peroxide from the same batch, this will ensure that even if the concentration is slightly different to that stated, each repeat will have an equal concentration.

Concentration of catalase (liver solution)- Because the concentration of catalase could be different in different parts of the liver, all of the repeats will be performed using the same batch of liver solution. The liver solution will be made to a concentration determined in the pilot experiment.

Volume of hydrogen peroxide, liver solution and aspirin solution- These need to be kept constant by measuring them accurately. They will be measured using syringes which should deliver accurate volumes within 1% of the reading.

Volume of oxygen produced in the reaction- This is measured using the manometer gauge, it can be measured to the nearest 0.5mm This gives an error of � 0.8 mm� for the volume of oxygen produced. Approximately 60mm� of oxygen is produced this gives a percentage error of around 1.3 %. This is very accurate.

Pilot

The aim of this pilot investigation is to find a concentration of hydrogen peroxide and concentration of liver which will cause the reaction to occur at a suitable rate.

The rate of the reaction must be fast enough to produce enough oxygen for it to be measurable, but must not be to fast as this would make it difficult to measure and the reaction would be completed too quickly. Hopefully the reaction will produce approximately 60mm� of oxygen in about 30 seconds, this would move the manometer bubble by 20mm. Increasing the concentrations of hydrogen peroxide and bovine liver catalase will increase the rate of reaction because there will be more collisions between the active sites of catalase and the hydrogen peroxide.

Method for pilot

The catalase solution is made by liquidising the liver with water. The first concentration I will attempt will be 100g of liver with 200ml of water, once processed it will be filtered through a funnel with filter paper (see fig.2 on page 8). If this concentration is not suitable other concentrations will be attempted until an appropriate concentration is found.

Hydrogen peroxide solutions will be made using various dilutions of 1 mol/dm� hydrogen peroxide. Each will be tested to the most suitable concentration.

To carry out the experiment measure 1ml of liver solution, and put it in the test tube. In the final experiment the liver solution will need to have 2cm� of each of the different aspirin solutions added to it and 2cm� of pH 7 buffer, but in this pilot I will add 4cm� of water (see fig.1 on page 8). The bung should be placed on the test tube, then 2cm� of the hydrogen peroxide should be added. The manometer is pushed into the bung the placement of the bubble is measured and timer is started. The manometer bubble should move as oxygen is produced by the reaction, the time taken for the bubble to move 20mm should be measured.

Results of Pilot

Table 1. Finding appropriate concentrations of liver and hydrogen peroxide

Volume of liver solution (ml)

Concentration of liver solution (%of original solution)

Volume of hydrogen peroxide solution (ml)

Concentration of Hydrogen peroxide (mol/dm� )

Volume of water (ml)

Notes

3

100

3

1

2

Reaction was much too fast and also frothed right up to the top making a mess. Volume of solutions needs to be reduced.

1

100

1

0.5

3

Reaction was completed very quickly(H2O2 used up), needs a greater concentration of H2O2

1

100

5

1.0

2

Too fast- should use more water to dilute the solution and dilute the liver solution, try using less H2O2 but still need more than previous attempt

1

14

1

1.0

4

Still much too fast- should dilute the liver further

1

0.7

1

1.0

5

Too slow- liver too dilute

1

1.4

1

1.0

4

Perfect!

In the final experiment the following volumes and concentrations of solutions will be used:

1ml of 1 mol/dm� Hydrogen peroxide

1ml of 1.4% Liver solution (made a batch in pilot)

2ml of Aspirin solution (varied concentrations)

2ml of pH 7.0 Buffer solution

Equipment

Test tubes

Test tube rack

Syringes

Beakers

Blender

Funnel

Muslin fabric

Manometer gauge (2mm diameter)

Bung (fits manometer and test tube)

Stopwatch

Gloves

Water bath (30�C)

Stirring rod

Chemicals/materials

1 mol/dm� Hydrogen peroxide

Calf liver

pH 7 Buffer

Aspirin tablets (300mg)

Distilled water

Accuracy of equipment

Syringes- These give a reading which is �1% of the actual volume, this is very accurate and make syringes a suitable piece of equipment for measuring out volumes of the different solutions used.

Manometer gauge- The manometer gauge can be read to the nearest 0.5 mm using the markings along it. This gives an error of � 0.8 mm� for the volume of oxygen produced. Approximately 60mm� of oxygen is produced this would give a percentage error of around 1.3 %. This is very accurate.

Stopwatch- This gives a time reading accurate to the nearest second, so the error is �0.5 seconds. Assuming that the reaction takes about 30 seconds this gives a percentage error of about 3%, this is quite accurate.

Method

(see (fig1 and 2) for further explanation )

1. Make a liver solution: Put some liver, approximately 100gm, in to the blender with 200ml of water, blend it until the mixture is smooth. Filter the liver solution through a funnel with muslin fabric to remove any solid bits. The liver solution must now be diluted, in the pilot run of this investigation a solution was mixed using 1ml of liver solution to 70ml of distilled water. The same batch must be used throughout the investigation. The concentrated liver solution may be disposed.

2. Making aspirin solutions: Measure out 30ml of distilled water using a syringe and add the appropriate number of soluble aspirin tablets (see table2). Use a glass stirring rod to ensure the solution is mixed properly.

Table 2. mixtures for aspirin solutions

Aspirin tablets

Total Aspirin (mg)

Volume of water (ml)

Concentration (gdm� �)

0

0

30

0

0.5

150

30

5

1

300

30

10

1.5

450

30

15

2

600

30

20

3. Measuring the rate of reaction: All solutions (in labelled beakers to avoid confusion) and glassware used must be kept in a 30�C water bath for this part of the experiment. (see fig. 1)

i. Measure out 1ml of diluted liver solution, 2ml of pH 7.0 buffer and 2ml of aspirin solution using three separate syringes and put these into the test tube. Shake gently to ensure the solutions are mixed and leave for 10 seconds. Put the bung on the test tube

ii. Measure out 1ml of the 1 mol/dm� hydrogen peroxide solution in a syringe. The hydrogen peroxide is added through the hole in the bung. The manometer must be added quickly after the hydrogen peroxide. Once the manometer bead is moving at a steady speed, because pushing the manometer into the bung causes some initial disturbance, a starting point for it’s movement must be noted and the timer started, when the bubble has moved 20 mm the timer must be stopped. The time taken for the bubble to move 20mm should be recorded in a results table.

iii. Repeat step 3 six times for each of the five aspirin concentrations

Repeats

There will be six repeats of each of the five aspirin concentrations, this will help to show whether the results are reliable or not. If the variables are controlled properly there should only be a small variation in the results. A large variation in the results shows that there is a problem with the method and that the results cannot be trusted to be accurate.

Risk Assessment

If all the precautions and procedures shown below are followed then the risks involved in performing this investigation are acceptable.

Source of risk

Safe handling

Emergency Procedures

Disposal

Hydrogen peroxide

(corrosive, harmful)

o Wear goggles

o Avoid skin contact

o Avoid ingestion

o Contact with eyes: rinse for at least ten minuets and seek immediate medical advice

o Contact with skin: wash off thoroughly with water seek medical advice if skin reddens

o Ingestion: drink plenty of water and seek immediate medical advice

With the low concentrations used in this experiment it acceptable to dispose of down the sink with plenty of water.

Calf liver (pathogens)

o Wear gloves

o Avoid skin contact

o Avoid ingestion

o Keep refrigerated when not in use

o Contact with skin: Wash using soap and water

o Ingestion: Seek medical advice

Liver solution may be flushed down the sink with water. Any remaining liver can be wrapped in a polythene bag before being disposed of by the bin.

Aspirin (toxic in high doses)

o Must be kept in a labelled container

o Avoid ingestion

o Ingestion(small quantities): inform teacher stating number of tablets ingested

o Ingestion(large quantities): seek medical advice immediately

Excess aspirin solution may be flushed down the sink.

Glassware

o Do not use chipped glassware

o Keep glassware in centre of bench to avoid breaking it.

o If glass is broken: Warn all people in the area, sweep broken glass into a dustpan and dispose of glass as soon as possible

o If someone is cut: Treat according to severity of the wound, for deep wounds seek medical advice.

Broken glass is disposed of into a special glass bin. It should not be put in a normal bin as anyone who handles the rubbish could get cut through the bin liner.

Diagrams for method

Fig. 1 Measuring the volume of oxygen produced by the catalase

Fig. 2 making liver solution

Results

Table 3. raw data-the time taken for the manometer bead to move 20mm for different aspirin concentrations.

Aspirin concentration (gdm� �)

Time taken for manometer bead to move 20mm (s)

0

24

26

25

26

25

24

5

30

29

30

28

28

30

10

33

32

35

32

35

34

15

37

37

36

38

38

38

20

38

38

39

40

42

42

Calculations

The volume of oxygen produced when the bead moves 20mm is ? x r2 x l, which is…. ? x 1 x 20 = 62.8 mm3 . To find a rate of reaction, which is in terms of the oxygen produced per second, divide the volume of oxygen produced (62.8 mm3) by the time taken.

Table 4. The rate of reactions for different aspirin concentrations.

Aspirin concentration (gdm� �)

Rate of reaction (in terms of oxygen produced) (mm3 s-1)

0

2.62

2.42

2.51

2.42

2.51

2.62

5

2.09

2.17

2.09

2.24

2.24

2.09

10

1.90

1.96

1.80

1.96

1.80

1.85

15

1.70

1.70

1.75

1.65

1.65

1.65

20

1.65

1.65

1.61

1.57

1.50

1.50

Table 5. Average and minimum and maximum values for rate of reaction for different aspirin concentrations.

Aspirin concentration (gdm� �)

Average rate of reaction (mm3 s-1)

Minimum rate of reaction (mm3 s-1)

Maximum rate of reaction (mm3 s-1)

0

2.52

2.42

2.62

5

2.16

2.09

2.24

10

1.88

1.80

1.96

15

1.68

1.65

1.75

20

1.58

1.50

1.65

Graph

Fig3: The rate of the breakdown of hydrogen peroxide with catalase, for different aspirin concentrations, in terms of the oxygen produced; error bars show the minimum and maximum values for each data set.

Analysis

The results and graph produced from this investigation show that aspirin reduces the rate of the bovine liver catalase-aided breakdown of hydrogen peroxide into oxygen and water. Increasing the concentration of aspirin results in greater inhibition of the reaction. This supports the hypothesis that aspirin is an inhibitor of bovine liver catalase.

Because nobody has fully investigated the enzyme inhibiting properties of aspirin, it is unknown whether the inhibition is competitive or non-competitive, or active site directed or non-active site directed. The aspirin could work by binding to, either permanently or temporarily, a part of the catalase enzyme and causing a change in shape of the active site. This would mean that the substrate (hydrogen peroxide) would not fit into the active site because its shape is no longer complimentary to it. The other way that the aspirin could work is by entering the active site itself, again this may be a permanent covalent bond or a temporarily caused by the inhibitor randomly moving into the active site, in either case the substrate cannot enter the active site because it is blocked by the inhibitor. With any of these possibilities increasing the concentration of aspirin will lead to more collisions between the aspirin and catalase enzyme, which will cause a greater percentage of the enzyme to be inhibited. This slows the rate of reaction because there are less active sites available for the hydrogen peroxide to bind to, this causes collisions with uninhibited active sites to be less frequent.

Increasing the concentration of aspirin made the most difference between 0gdm-3 and 10gm-3 where the rate of reaction fell by 0.64mm3s-1, between these points the relationship between aspirin concentration and rate of reaction is shown on the graph as an almost straight line (see fig 3). After this point the change in rate of reaction compared to the increase of aspirin concentration began to reduce. This is because the percentage increase in aspirin concentration is smaller so between 5gdm-3 and 10gdm-3 the concentration is doubled, but to double the concentration again it must be increased to 20gdm-3. This is shown in the results because the change in rate of reaction between 5gdm-3 and 10gdm-3 is 0.28 mm3s-1 and between 10gdm-3 and 20gdm-3 the rate of reaction fell by 0.30mm3s-1. This trend is shown graphically as a curve (see fig 3).

Eventually if the aspirin concentration was increased further, the graph would probably level off as it reached the maximum inhibition that can be caused, however, this did not happen at the concentrations used in this investigation. This is reassuring because it means that although aspirin will affect the metabolic processes in your body, the concentrations taken by people will not have too great an effect; remember that although concentrations used in this investigation were based on the number of tablets that people may use, they were diluted much less than they would be in the human body

The shape of the graph (see fig. 3) suggests that the curve would level off before the rate of the breakdown of hydrogen peroxide is zero, this would mean that at the maximum inhibition the reaction would still occur. If this is true it suggests that aspirin may inhibit the enzyme by bonding to it in a way that alters the shape of the active site making it less of a good fit for the substrate, this way even though the enzyme is being inhibited by the aspirin, it may breakdown some of the hydrogen peroxide molecules that come into contact with it. However, this would have to be investigated further before any conclusions about the action of aspirin could be made.

Perhaps this investigation also spreads some light over the claims that taking aspirin regularly can reduce the risk of cancer, it could be that by inhibiting some of the enzymes involved in metabolism, that aspirin slows the rate of growth in cells so reduces the spread of cancerous cells. However, until more research has been made we cannot be sure.

Inconsistencies in the data

The main problem with the data is that some of the error bars are quite close to each other with one overlap between the minimum value for 15gdm-3 of aspirin and the maximum value for 20gdm-3 of aspirin (1.65mm3s-1 of oxygen produced). However, because there is a clear overall reduction and no overlaps in the rest of the data, it may be assumed that the data is beginning to overlap because the change in rate of reaction is slowing down, it may be that if higher concentrations of aspirin were used the graph would level off soon after this point.

There have been no values that have been considered anomalous as all of the values for rate of reaction are within 0.1 mm3s-1 of their average.

Evaluation

Variability of the results

Results from this investigation were not very variable with the largest standard deviation in rate of reaction being 0.09 mm3s-1. The problem, however, is that the difference in rate of reaction caused by the aspirin is quite small, this is why the error bars show some very close minimum and maximum values with one overlap. The actual values for rate of reaction seem to be quite precise because their percentage errors (based on standard deviation) are relatively small between � 2.2% and � 4.6%.

But when the difference between results is quite small, for example between aspirin concentrations of 15gdm-3 and 20gdm-3, where the difference in rate of reaction is -0.10, the percentage difference is -6%, so a variability of � 4.6% in the results for 20gdm-3 means that these results cannot be used to prove that there is a difference in rate of reaction between aspirin concentrations of 15gdm-3 and 20gdm-3. However, because the overall reduction between an aspirin concentration 0gdm-3 and 20 gdm-3 is large compared to the standard deviations, with an overall reduction of nearly 1mm3s-1 or percentage reduction of 37%, it is quite safe to suggest tentatively that aspirin inhibits the action of bovine liver catalase.

Limitations of the technique

The main problem experienced with this technique was that the movement of the manometer bead was never steady. The bead would stay still for a while and then jump to the next point, this is probably the main reason for variability within the results. The reason that this occurred is probably because there needed to be some pressure to make the bead move, so pressure had to build up and then would be released in one go. This is likely to be caused by surface tension in the bead, which made of a small drop of water. This produces variability in the results because depending on whether the bead was stationary or moving as it reached the 20mm displacement, there may be a period of time where the bead is nearly there and pressure must build up before it moves again, or may move immediately. Another potential problem with the technique is that only a small amount of gas is produced before the measurement is made, by having a greater amount of gas produced any small inconsistencies in the rate of reaction are evened out.

This may be part of the solution to the problem of the uneven movement of the bead, because if it had further to move then the difference made by whether it is moving or stationary as it reaches the final displacement, would be small in comparison to the overall time. Another area where error may occur is the measurements of the different solutions. Although the syringes used were very accurate, an even greater accuracy could be obtained if a larger volume of the solutions is measured out, as the larger the volume measured out is, the smaller percentage error it has.

Improvements

There are two main improvements that can be made to the technique of this experiment which would help to gain more accurate and conclusive results.

Instead of using a manometer to measure the oxygen produced, a gas syringe would be used. A gas syringe measures up to 100ml of a gas, and has markings for every mm of displacement. The large volume of gas produced would help to even out any inconsistencies, and because the enzyme catalase is such a fast acting enzyme using it at a greater concentration means that producing large volumes of oxygen is quite realistic. The use of a gas syringe may also help to stop the inconsistencies of movement observed while using the manometer because it is designed to have very little friction in its movement. It also means that larger volumes of the catalase and hydrogen peroxide solutions can be used, as the gas syringe will be able to measure the larger volumes of oxygen produced.

Use larger volumes of the solutions; this increases the accuracy of the measurements made for the volumes of solution because the error becomes smaller in comparison to the actual measure made. It also means more oxygen can be produced making the measurement for oxygen production more accurate. If larger volumes of the solutions were used in the original experiment, they bubbled up the test tube into the manometer, however, if a conical flask was used the volumes of the solutions used could be increased greatly.

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