Concentration of Enzymes
The amount of enzymes affects the rate of reaction. The enzyme molecules bind with the substrate molecule to lower the activation energy of the substrate. If there are more enzyme molecules present then substrate molecules there would be more collisions between the enzymes and the substrates. Increased chances of there being a collision would increase the rate of reaction, when the number of enzyme molecules exceeds the number of substrate molecules then the rate of reaction will not increase further.
Concentration of Inhibitor
If the concentration of the inhibitor is high then a higher number of inhibitor molecules can bind with the active site of the enzyme, rendering the enzyme ineffective, which will in turn reduce the rate of reaction. If the concentration of the inhibitor was lowered then there will be less of the inhibitor molecules inhibiting the enzymes, therefore there will be more unaffected enzyme molecule to bind with the substrate.
pH
pH is a measurement of the concentration of hydrogen ions in a solution, the lower the pH the higher the hydrogen ion concentration. Hydrogen ions can interact with the R groups of amino acids, affecting the way in which they bond with each other and therefore affect their 3D arrangement. Most enzymes work faster at pH7 which is neutral. If a different pH is used the enzymes will be affected and the rate of reaction will change
Apparatus
The apparatus that are to be used in the experiment should be chosen very carefully as the appropriate apparatus should be used to provide the optimum accuracy from the results.
The list of apparatus that is to be used is written here.
1 x Retort stand
1 x Side arm test tube
1 x Delivery tube
1 x Rubber bung
1 x Water bath
1 x Petri dish
4 x Measuring cylinder (50 cm³)
1 x Scalpel
1 x Ceramic tile
1 x Forceps
1 x Liver
1 x Electronic scale
1 x Distilled water
1 x Copper (II) Sulphate solution (1 mol)
1 x Hydrogen peroxide solution (1 mol)
1 x Stopwatch
1 x Beaker
The reasons as to why these apparatus were chosen are listed below.
Retort stand – a retort stand is needed to hold the test tube safely in place.
Side arm test rube – this was chosen because the side arm test tube is ideal for this experiment. Oxygen needs to be collected without it escaping; the oxygen can exit through the side arm, which cannot be performed in a regular test tube.
Delivery tube – once the oxygen leaves through the side arm of the tube it has to be guided to the cylinder where it will be collected. The delivering tube is perfect for this.
Rubber bung – once the reaction has started it is imperative that the oxygen doesn’t escape. That is why the rubber bung is chosen as it can create an airtight seal on the test tube forcing the oxygen to leave through the side arm.
Water bath – the temperature will be a fixed variable through out the experiment, this can be achieved with the aid of the water bath. The test tube would be placed in the water bath while the reaction takes place.
Petri dish – the petri dish was chosen because it is ideal shape to hold the measuring cylinder and water.
Measuring cylinder – four measuring cylinders are required for the experiment. The first three are used to measure the correct amount of distilled water, copper (II) sulphate solution and hydrogen peroxide solution in individual cylinders.
Scalpel – a scalpel is preferred to an ordinary knife as the cutting blade is sharper and far more accurate. An accurate blade is needed to cut a small piece of liver.
Ceramic tile – the ceramic tile is used to cut the liver, this prevents the worktable from getting damaged.
Forceps – this is used to place the piece of liver in the test tube.
Liver – the liver contains the enzyme called catalase. The liver is easy to prepare and to work with.
Electronic scale – this is used to measure out the correct amount of liver. This is used, as it is a very accurate form of weighing objects when compared to a mechanical one.
Distilled water – the distilled water is used to reduce the concentration of the copper(II)sulphate solution. Distilled water is ideal for this, as it doesn’t contain any other substances as opposed to tap water.
Copper (II) sulphate solution – the copper in copper (II) sulphate acts as the inhibitor for the reaction. That was why this chemical was chosen.
Hydrogen peroxide solution – this was chosen as it reacts with the catalase to produce water and oxygen. The amount of oxygen produced will determine how fast the reaction is happening
Beaker – the copper (II) sulphate solution and distilled water would be mixed in the beaker.
Once the apparatus are chosen they should be set up in a way that the experiment could be performed at ease and reduce any chanced of receiving anomalous results.
Diagram 2 shows how the apparatus should be set up.
Diagram 2
Diagram of the experiment
Method
An appropriate method should be devised to which would produce the best possible results and minimize any possible errors.
Once the method is devised, preliminary tests should be carried out to firstly see if the method works and secondly to determine a suitable range in the concentration of the inhibitor that would produce a wide range of results.
The method is described in the following paragraphs in simple steps
Step 1
The apparatus will be set up as shown in the diagram 2. Then using a scalpel (care is needed here to ensure safe working) cut a piece of liver weighing at about 0.1grams to 0.2grams, will be cut on the ceramic tile.
Step 2
Then using the forceps the piece of liver should be placed in the bottom of the side arm test tube ensuring that the liver does not touch the sides of the test tube.
Step 3
The concentration of the copper(II)sulphate should be diluted according to Table 1 on page 7 (in some cases the dilution of copper(II)sulphate is not required). The table specifies the amount of distilled water and copper(II)sulphate that should be mixed together in a beaker to achieve the desired concentration. Once the copper 2 sulphate has been diluted it should be poured into the test tube containing the liver.
Step 4
Measure out 5cm³ of hydrogen peroxide in a measuring cylinder. And pour the whole contents of the measuring cylinder into the test tube containing the liver and the diluted copper(II)sulphate.
Step 5
Place the bung immediately, making sure that an airtight seal is achieved. Then time the reaction for 4 minutes making note of how much oxygen is collected at the end of the 4 minutes. The experiment will be performed at a constant temperature of 30°C, this will be achieved with the use of the water bath.
Step 6
The apparatus should be thoroughly cleaned with distilled water before they can be set up again. It is necessary that the experiment be repeated for a second and third time. After that calculate the average volume of oxygen collected.
Table 1- A table to show which experiments are to be carried out
Fair Test
To achieve accurate and reliable results, a fair test must be carried out. Even a slight change could alter the outcome of a test, so devising a fair test is imperative.
To make the experiment a fair test, firstly the temperature at which the reaction takes place in must remain constant throughout. To do this a water bath would be used. It is vital that the temperature remains constant because enzyme activity increases or decreases depending on whether the temperature is high or low. At low temperatures the reactions take place at a low rate.
The reason is that the substrate molecules and the enzyme molecule are moving relatively slowly due to the fact that they contain less energy as a direct result of the low temperature. This would mean that the enzyme molecules and substrate molecules would collide less often. Therefore the binding between enzyme and substrate reduces hence a slower reaction. If the temperature were to be increased the enzyme and substrate molecules would move faster, more collisions would take place and as they collide they would collide with more energy helping the bonds to break faster, this all adds up to a faster reaction.
If one experiment was done at 35°C and another was done at 20°C then there will be more enzyme activity in the former rather than the latter.
Secondly the volume of hydrogen peroxide and the total volume in the test tub must be the same in all the experiments.
And lastly all the equipments should be washed properly after each test with distilled water to reduce the risk of contaminating the next test.
Preliminary Work
As I have mentioned earlier the preliminary work is carried out to firstly see if the method works and secondly to determine a suitable range in the concentration of the inhibitor that would produce a wide range of results. Also this opportunity could be used to modify the method or the way the equipments are used.
Apparatus
For the preliminary work these apparatus were used-
1 x Retort stand
1 x Side arm test tube
1 x Delivery tube
1 x Rubber bung
1 x Water bath
1 x Petri dish
4 x Measuring cylinder (50 cm³)
1 x Scalpel
1 x Ceramic tile
1 x Forceps
1 x Liver
1 x Electronic scale
1 x Distilled water
1 x Copper (II) Sulphate solution (1 mol)
1 x Hydrogen peroxide solution (1 mol)
1 x Stopwatch
1 x Beaker
For the preliminary work three tests were carried out the determine the range of the variable, the concentration of the inhibitor (copper(II)sulphate). The apparatus was set up in the way that was explained in the method and the test carries out as said in the method. Here are the results.
The three tests were chosen so that the top end of the range could be established as well as the lower range. The lower range was given by the first test as it contained the highest possible concentration of copper(II)sulphate that would have been used in the real experiment. This was a 1 molar solution. An opposite approach was used to find the top range. The top range was given by the last of the 3 tests. This test had no traces of copper(II)sulphate at all, which means there was no inhibition and the rate of reaction would be the highest out of the three tests.
A range of 71cm³ in the amount of oxygen collected seems sufficient enough to perform tests which produce results with a wide range.
A problem was encountered while performing the preliminary work. The measuring cylinder that was described in the method had a maximum capacity of 50cm³. The purpose of his measuring cylinder was to collect the oxygen that was given out during the reaction.
During the third experiment the amount of oxygen being released had exceeded the maximum capacity of the measuring cylinder, to compensate for this a measuring cylinder with a larger capacity (100 cm³) was used. This ensured that all the oxygen was being collected.
Apart from that there would not be any other changes to the equipment or the method.
Risk Assessment
There are always risks involved when handling any kind of chemicals. Both the copper(II)sulphate and Hydrogen peroxide are irritants. To ensure that there is maximum safety, all personnel involved in the experiment should wear proper eye protection, in the form of goggles and as well as gloves to protect the hands.
Not only should they take care in handling the chemicals if there are any spillages then the person responsible should clean up the chemicals, so that it is not left for others to find.
If a person does come in contact with hydrogen peroxide on the skin then plenty of water should be applied to the effected part. If the chemical come in contact with the eye, immediately rinse the eye with water, and seek medical attention straight away. If the chemical is swallowed the drink plenty of water and seek medical attention straight away.
If inhaled lithium hydroxide can cause severe irritation and corrosive tissue damage, similar to other strong bases. Like sodium hydroxide and potassium hydroxide. If inhalation of lithium hydroxide occurs, remove source of contamination or move victim to fresh air and seek medical attention.
Copper(II)sulphate equal to and greater than 1 mole is harmful. It may be irritating to the eyes and skin
Copper(II)sulphate can cause severe irritation and corrosive damage to eyes. If there is contact with eyes, gently blot or brush away dust quickly. Immediately flush contaminated eye run with water, seek medical attention. To prevent this from happening wear appropriate eye protection.
Copper(II)sulphate has been known to sensitise the skin.
Table 2 - A table to show the volume of oxygen given off at various concentrations of
copper(II)Sulphate.
Analysis
The results of the experiment was gathered and a series of graphs were produced to help interpret the information more precisely.
The results of the experiment show that as the concentration of copper(II)sulphate increases, the rate of enzyme controlled reaction decreases. This happened because the copper in copper(II)sulphate was binding with the enzyme molecule thus preventing the enzyme from functioning properly. This would mean that the activation energy would not be lowered sufficiently enough, so the reaction would be slower as a result.
The maximum rate of reaction measured was in the controlled experiment. In this tube there was no copper(II)sulphate, only catalase and hydrogen peroxide. The amount of oxygen released was 79cm³, but when you compare this with the amount of oxygen released during the last test which had a 1 molar solution of copper(II)sulphate there is a massive difference. The amount of oxygen released in that particular test was 10 .5. That is a decrease of 91.7% in the amount of oxygen released. This is strong evidence to support the idea that copper is in fact an inhibitor of catalase.
The gradient between 0mol and 0.2mol was recorded at 80 and the gradient between 0.2 mol and 0.4 mol was recorded at 80. This indicates that there was a sharp decline in the rate of reaction when the concentration of the copper(II)Sulphate was increased from 0mols to 0.4. After that the rate of reaction increases briefly but then sharply declines again. (refer to graph 3 for more details)
By carrying out the experiment that I have done I have come to a conclusion that copper sulphate is an inhibitor.
Graph 1
Graph 1shows all the different dilutions with the copper(II)sulphate. If you refer to the graph you will see that as the concentration of the copper(II)sulphate is increased the rate of reaction decreases and also the amount of oxygen released is decreased. This happens because the copper in copper(II)sulphate was binding with the enzyme molecule, preventing the enzyme from functioning properly.
Graph 3
This shows the gradients of the results and from that the rate of reaction can be worked out. This is shown in graph 3. As you can see the rate of reaction slowly declines as the concentration of copper(II)Sulphate is increased, but the rate of reaction increases and decreases in between results.
The gradient goes from 80 to 50 in between 0.2 and 0.4 mols but then the rate of reaction decreases significantly at 0.4mols to1.6mols when compared to the gradient from 0.2 to – 0.4mol. This however has no bearing on the final results, as there are no anomalous results.
There do not seem to be any anomalous results, the probable reason as to why there were no anomalous results would be that all the procedures explained in the method were observed. The temperature at which the experiment was carried out remained constant with the aid of a water bath, this meant that the rate of reaction would not increase as a direct result of the increase in temperature or the loss of temperature which would result in a slower reaction. It was suggested that the experiment should be performed at room temperature but that would have been a mistake as the temperature of a room doesn’t always stay constant. This would have meant there would have been incorrect results.
The liver was measured by an electronic scale, this would have definitely have contributed to the fact that there were no anomalous results. The accuracy provided by the electronic scale meant that the liver could be measured to three decimal places, this provided a smaller margin of error.
Other factors that also contributed to the fact that there were no anomalous results present were the fact that all the equipment were washed with distilled water to remove dirt and other unwanted particles. If the dirt had not been removed the reaction between the particles could have been affected. This could have given false results.
My final conclusion for my experiment would be that copper in copper(II)sulphate is an inhibitor of catalase. The reason is that if copper wasn’t an inhibitor of catalase the amount of oxygen released would have been similar in all the tests regardless of the concentration of copper(II)sulphate that was used, as it would not have any affect on the reaction. But that was not the case, in fact it was quite the opposite. The amount of oxygen released started to decrease as the concentration of copper(II)sulphate increased, this would indicate that the rate of reaction would decrease too. The rate of reaction would only decrease if the copper in copper(II)sulphate was inhibiting the catalytic functions the enzyme. The copper is inhibiting the enzyme by binding with it and rendering it useless.
There are two types of inhibitors, non-competitive and competitive. Competitive inhibitors have competition to content with as they bind only briefly. The competition is in the form of the substrate. If there is more of the substrate than the inhibitor then the enzymes functions would be unaffected. Non-competitive inhibitors remain permanently bonded with the enzyme there fore permanently stop the enzyme from ever functioning. The concentration of substrate has no bearing of the inhibition.
I have come to the conclusion that the copper in copper(II)sulphate is a non competitive inhibitor. The reason I have come to this conclusion is that as I have mentioned earlier in competitive inhibitors if there is more substrate present than the amount of inhibitor, the substrate molecule can easily bind to the active site in the usual way which means the enzymes function remains unaffected. In four out of the six tests there was more substrate present than copper(II)sulphate, but in all four of the tests the rate of reaction was reduced, which meant that the copper was inhibiting the function of the enzyme. In the second test the there was 5cm³ of hydrogen peroxide and 1 cm³ of copper(II)sulphate. If copper was a competitive inhibitor there would have been no inhibition, but there was 13.5 cm³ of less oxygen being released from the reaction. Theses are not the characteristics of a competitive inhibitor. Therefore copper must be a non-competitive inhibitor.
.
Evaluation
The results obtained from the experiment was accurate enough to be able to find out that copper is a non competitive inhibitor of catalase. But I feel that the results could have been more accurate. The main reasons that I raise this matter are that there were a few flaws in the method that had been used. Firstly as the hydrogen peroxide was added in the test tube the rubber bung had to be replaced very quickly, during that split second oxygen could have escaped. Whether the amount of oxygen that escaped is significant enough to give wrong results is not known, but the possibility still remains there.
Also there could have been errors made when the hydrogen peroxide, copper(II)sulphate and distilled water were being measured in the measuring cylinder. These mistakes were most likely human error. The error would most likely have been a misjudgement of just exactly how much was in the measuring cylinder as a minuscule usually forms. This confuses the mind, which leads to the wrong amount being measured. There is no definite solution to this problem, the only logical thing to do if not sure is to get a second opinion to confirm the initial opinion.
I think that my results are reliable enough to support my conclusion.