Enzymes: -
There are two main enzymes that are used in anaerobic respiration, decarboxylase and dehydrogenase. Enzymes and substrate molecules are constantly moving and often collide. When the enzymes collide the substrate may fit in to the enzymes active site for a brief moment, forming an enzyme substrate complex. Once the substrate enters the active site of the enzyme, the enzyme changes shape to mould itself around the substrate. This is called induced-fit. While this is happening the R group on the polypeptide forming the active site are brought in to a position which combines with the substrate, forming temporary bonds. The bonds within a single substrate may be put under stress so that it is broken down. The reactions then form a product(s) and then leave the active site leaving it free to react with another substrate molecule.
From the AS level I found out that PH, Substrate concentration Enzyme concentration and Temperature are all factor that effect enzymes. The factor that will affect my experiment is the temperature on the dehydrogenase enzyme. At a low temperature the molecules should have a slow reaction because there is little kinetic energy and the particles rarely collide. As the temperature increases there should be more collisions. The enzyme then reaches its optimum temperature; this is where the rate should be at its fastest. The enzymes then start to loose their tertiary structure and the bonds that hold the polypeptides in their specific shape are then broken, changing the shape of the active site. The active site shape has now changed the substrate cannot fit, therefore the enzyme is said to be denatured.
When the temperature has increased most of the enzymes are denatured but not all of them. If all the enzymes have not been denatured then there will still be a small reaction-taking place but using an enzyme inhibitor can stop this. There are two types of inhibitors, Competitive and Non-Competitive Inhibitors.
Competitive Inhibitors: -
These are molecules that enter the active site. These molecules have a similar shape to the substrate molecules so they fit into the active site. When this happens the substrate cannot fit into the active site therefore the enzyme cannot catalyse the reaction. Inhibitors and substrate molecules cannot enter the active site at the same time. Inhibitors are constantly moving in and out of active sites therefore adding more substrate can reduce the effect of an inhibitor.
Non-Competitive Inhibitors: -
These do not occupy the active site but they find another site on the enzyme molecule. This then changes the shape of the active site so the substrate molecule cannot fit anymore. If the concentration of this type of inhibitor is increased then the reaction can be slowed to zero but if the concentration of the substrate is increased then there is no effect on this type of inhibition.
Both competitive and non-competitive inhibitors are both reversible inhibitors but there are irreversible inhibitors. These are molecules that permanently bind themselves to the enzyme molecule. This then effects the enzyme concentration lowering the rate of reaction slowly to zero.
Prediction: -
I predict that as I increase the temperature the rate of respiration increases therefore as I increase the temperature the time taken for the TTC solution to change from colourless to pink will decrease. At 25oC I predict that the reaction will be slow and the time that it takes for the TTC to change from colourless to pink will be long. This is because the enzymes are working very slowly and there is very little kinetic energy there fore there aren’t many enzyme substrate reactions. At 30oC I predict that the time taken for the TTC solution to change from colourless to pink will still be slow but faster than it was at 25oC. This will be the same up to about 45oC. At 45-50oC I predict that the rate of reaction will be high and the enzymes will be working very fast and also denaturing therefore the TTC solution will turn from colourless to pink quickly.
I can support this prediction with knowledge from AS and A2 biology. From the AS I learnt that as I increase the temperature the enzymes will work faster and there will be more successful collisions between the enzyme and substrate molecules. The rate of reaction will increase as I increase the temperature until a point where the optimium temperature is reached (point at which the enzymes work their best) and the reaction starts to drop. This is because as I increase the temperature the heat will break the hydrogen, ionic and disulphide bonds, the tertiary structure then collapses and the shape of the active site changes, the substrate molecule cannot fit and the substrate can no longer be catalysed. This is called denaturation. I predict this to happen at about 40oC, as this is the optimum temperature at which the dehydrogenase enzyme works. As the dehydrogenase enzyme works faster it removes more hydrogen from the metabolic pathway for example in glycolosis where NAD is reduced, the TTC picks up the hydrogen and turns the TTC solution colourless to pink. The enzyme responsible for this is dehydrogenase. When I get my results I will manipulate the data to find the rate of reaction by dividing 1 over the time to find the rate of reaction. I will then make two graphs, one with the average time against the temperature and the rate of reaction against the temperature. This is what I predict them to look like.
Rate of Reaction (1/Time) against Temperature: -
I predict that this graph will show that as I increase the temperature the rate of reaction increases to a point where the enzymes denature so the reaction slows and eventually drops.
Average Time against Temperature: -
I predict that this graph will show that as I increase the temperature, the time taken for the TTC solution to turn from colourless to pink decreases. This is because the enzymes work faster. When the enzymes start to denature the time taken for the TTC solution to change colour starts to increase therefore the line begins to rise again. There will then be a point where the TTC does not change colour any further because the enzymes needed for the reaction to take place have denatured.
Although this is what I predict my graph to look like there is another factor that will affect the way it looks this is called Q10. This is something that takes place in enzymes when the temperature is altered. The theory of Q10 states that at every 10oC rise half the enzymes have denatured but the other half are working at double the rate therefore keeping the rate of reaction steady. Therefore Q10 = 2. This will influence my graph because it will take longer before the graph shows a drop in the rate of reaction. To get a drop in the rate of reaction to show on my graph I may have to do a larger range of temperatures.
If Q10 Takes place then this will change the shape of my graph showing the following: -
Rate of Reaction (1/Time) against Temperature: -
I predict that this graph will rise and at every 10oC the rate of reaction will double making the graph steeper until it shows a sign of reaching the optimum temperature.
Average Time against Temperature: -
I predict that this graph will drop because the time taken for the yeast suspension to change colour will decrease as the temperature is increased. The time taken for the colour change will be double as fast after the first 10oC because of Q10. The enzymes then start to denature and the line will show signs of rising as the time taken for the yeast suspension to change colour will rise again.
List of equipment: -
The equipment that I am going to use is: -
- Test tubes (x18)
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TTC solution (9cm3)
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Yeast Suspension (162cm3)
- Stop clock
- Bunsen burner
- Polystyrene cup
- Thermometer
- Test tube rack.
- Colorimeter
- Syringe
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Distilled Water (5cm3)
Method: -
Before I start my experiment I will draw a table to put my results on which will look like this:-
Once I have drawn the table I am going to carry out the experiment. First I am going to collect some test tubes and then put them into a test tube rack. I will then fill each one with 9cm3of the yeast suspension with one syringe and then keep this syringe separate away from the rest of my equipment especially the syringe used to pour the TTC in to the test tube to avoid cross contamination. I will then take a Bunsen burner, metal gauze and a tripod and then place a beaker of water with a thermometer inside it and then warm the water to the required temperature. While the water is heating I will use a different syringe and take 0.5cm3 of the TTC solution. Once my water is at the required temperature I will pour it into a polystyrene cup and fill it about a centimetre from the top. I will then place the thermometer inside the polystyrene cup. Then I will pour my yeast in to the test tube and place it in to the beaker to equilibrate for 5 minutes so that temperature of the yeast suspension reaches the required temperature then I will add the 0.5cm3 of the TTC solution and place a rubber bung on top of the test tube. Once the rubber bung is on the test tube I will shake it by turning it upside down 10 times so the TTC and the yeast mix together. I will then remove the rubber bung so the test tube does not shatter due to the collection of carbon dioxide from the respiration process. Then I will insert the test tube into the polystyrene cup that contains the water and thermometer. Then I will start the stop clock. While the experiment is taking place I will observe the temperature of the water if there is a drop, even though I am using a polystyrene cup as an insulator, there will still be a temperature drop when the experiment takes long for example at 25oC. To control this temperature drop I am going to add hot water to the polystyrene cup and also stir the water to mix it up. If the experiment lasts more than 2minutes I am going to shake the test tube to mix the TTC and the yeast as the yeast also separates in to two layers (one heavy and one light). Once the TTC has changed to pink I will stop the stop clock and record the time in seconds. I have to also remember the shade of pink by memory as it will continue changing colour, as all of the enzymes have not denatured. Once I have done this I will add an enzyme inhibitor called Urethane or Cyanide to my TTC-Yeast mixture to denature all of the enzymes. Once I have done this I will put it in a separate test tube and dilute it with a constant amount of distilled water to put in the colorimeter and measure the percentage absorbance of light. I will use the colorimeter by restarting the colorimeter using distilled water (5:5cm3) then I will take the blue-green filter and place it in the filter hole as this is the complimentary colour to pink. Once I have placed the filter in I will place some of my diluted TTC-Yeast mixture and then put it in to the hole provided for the dilute the solution and then test it and then note the percentage absorbance down. I have to dilute the TTC-Yeast mixture because it is too thick and the colorimeter would read 0% absorbance.
I will then record my percentage absorbance values in a table that will look like this
Once I have noted the absorbance I will do the experiment again but this time with another temperature and repeat it all three times and take an average and also find the rate of reaction. Before I start to take down results I will do a pulmonary experiment to test that every thing is working well and then I will start the real test.
Diagram: -
Variables: -
There are three types of variables: - 1. Independent variables (what I can’t control)
2. Controlled variables (what I can control)
3. Changing variable (what I am changing)
Independent variables: -
The things that I can’t control are: -
- The yeast activity. (This will affect my experiment because if the yeast is active the reaction will take place faster but if it is not active it is slow as the removal of hydrogen from the pathway takes longer).
Controlled variables: -
The things that I can control are: -
-
The volume of TTC. (This has to be controlled because if there is more TTC, the taken fore the TTC solution to change from colourless to pink increases this is because there is more of it to change colour therefore more hydrogen from the metabolic pathway is needed to be passed on to the TTC. I am going to control this by using a 0-1.0 cm3 syringe because it is accurate and precise compared to something like a measuring cylinder because it is to big where as a syringe is small and easier to adjust to make the amount of TTC exactly 0.5cm3).
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The volume of yeast. (This has to be controlled because if there is more yeast then the TTC solution will turn from colourless to pink more quickly. This is because the rate of respiration increases therefore more hydrogen is passed on to the TTC changing the colour of the TTC quicker. I am going to control this by using a 0-10.0 cm3 syringe to accurately take and insert a constant amount of yeast in to the test tube).
- The volume of water. (This has to be controlled because if the level water changers fore example for 1 test the level of water is below the level of TTC-yeast mixture and in another test the level of water it is over then this will effect the time taken for the TTC-yeast mixture to change its temperature and this will effect the time taken for the TTC solution to change colour from colourless to pink because if the level of water was above the level of the TTC-yeast mixture and if it heated up quicker than this will have an advantage as it will be hotter and the rate of reaction will be faster because the enzymes will be working faster. I will control this by adding a constant amount of water at a level that is above the level of the TTC-yeast mixture).
- The volume of glucose/sucrose. (This has to be controlled because this acts as the substrate in respiration as this is what is broken down in to either lactate (aerobic respiration) or ethanol (aerobic respiration). If there is more substrate to be broken down then more hydrogen and carbon dioxide is given off in the metabolic pathway hence turning the TTC solution pink more quickly. The glucose/sucrose has already been added to the yeast and has been controlled by adding a constant amount of glucose/sucrose to the yeast).
- The number of times that I shake the test tube. (This has to be controlled because the shacking of the test tube activates the yeast. If I shake the yeast it becomes more active thus it will give off more hydrogen, which will affect the time taken for the colour of the TTC to change. I will control this by shacking the test tube before the experiment and every 60 seconds by placing the bung on the test tube and shake it 10 times by turning it upside down. I will then remove the rubber bung).
- The concentration of TTC (This has to be controlled because if the concentration of the TTC solution is high the colour change would occur more quickly therefore to keep it a fair test I am going to keep the concentration of the TTC solution constant, 0.5%. I will control this by using the same TTC solution through out the whole experiment).
- The time left for the equipment to equilibrate (This has to be controlled because I change the time that I leave my equipment to equilibrate then it will effect the temperature of my experiment at the start for example if at one test I left the yeast with out the TTC to equilibrate for 5 minutes and at another test for 2 minutes then the test left for 5 minutes will have had a better chance of reaching the temperature required for the experiment. I will control this by leaving a constant time at the start of the experiment for the equipment to equilibrate).
- The batch of yeast (This has to be controlled because different yeast batches have different activities. This will affect my experiment because if 1 batch of yeast was active then rate of reaction would be faster and if another batch was not very active then my rate of reaction will be slow giving me inaccurate results. I will control this by trying to complete my experiment in one lesson so I can use the same yeast each time).
- Whether the yeast has been oxygenated (This has to be controlled because if the yeast has been oxygenated then the type of respiration that will take place is aerobic respiration and switch to anaerobic respiration later on in the experiment but if the yeast hasn’t been oxygenated then anaerobic respiration will take place not long after the experiment has started and this will effect the rate of reaction. I will control this by bubbling air into the yeast suspension).
- The time left for the yeast to be oxygenated (This has to be controlled because if the yeast is not oxygenated for long then the type of respiration could switch with out us noticing in one experiment and stay the same in another, this will effect the results. I will control this by leaving air to be bubbled in to the yeast suspension for a constant period of time).
- The shade of pink of my experiment (This has to be controlled because if the end point has changed to different shades of pink then this means that an increased or decreased rate of respiration has taken place).
Changing Variable:
- The temperature (This has to be changed, as this is what I am testing to see its effect on the respiration of yeast. I am going to vary this by using a Bunsen burner to change the temperature of the water and a polystyrene cup to insulate the heat. If the temperature starts to drop then I will keep adding hot water to try and keep the temperature constant although this method is very inaccurate).
Risk Assessment: -
Reason for choice of equipment: -
The reasons for choice of my method are: -
- Test tubes (x18)
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TTC solution (9cm3): -
This is an indicator that changes colour from colourless to pink when hydrogen is added. I am using this because when the yeast respires aerobically and anaerobically, hydrogen and carbon dioxide is removed from the metabolic pathway and picked up by the TTC solution, which then changes colour. As more hydrogen is added the shade of pink becomes darker. I have chosen to use 0.5cm3 of the TTC solution because this will change colour very quickly even if the yeast is not very active it wont take long therefore I get time for more repetitions to make my results more accurate.
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Yeast Suspension (162cm3): -
This is what I am going to use as the respiratory substrate for the experiment. Glucose and Sucrose may already be added to the yeast suspension. I need this because I have to add it to the yeast enable for it to respire as this is a vital part of respiration, aerobic and anaerobic. I have chosen to use 9cm3 of the yeast suspension because if the yeast is not very active then I still have a lot of yeast in my experiment to pass on hydrogen to only 0.5cm3 TTC. This will then speed up my experiment. I do not wish to use more than 9cm3 because the test tube is not that big and I need space to shake the mixture.
I am going to use the stop clock to measure the time it takes for the TTC solution to go from colourless to pink in seconds.
I am going to use this to heat the water for the water bath to the required temperature. A Bunsen burner is not very accurate compared to an electronic water bath as the temperature of the water can increase to high or decrease to low.
I am going to use this as a water bath for heating my solution to the temperature that I want and then I can add the test tube into the water bath and time the colour change. I chose a polystyrene cup as a water bath because they are good insulators and will reduce heat loss so I can try and keep the water at a steady temperature. Using a polystyrene cup is accurate and reliable as it keeps the temperature constant for longer.
I am going to use this to measure the temperature and also make sure that the temperature is constant.
- Test tube rack.
- Colorimeter: -
I am going to use this to find the percentage absorbance of light at the end of each test to find the end point of the experiment. This is the point where I stop the stop clock because I think from judging from my eye that the TTC-yeast mixture has reached the required shade of pink. I will then test the percentage absorbance of light to see if the shade of pink is constant at the end of each experiment. This will help increase the reliability of my results.
I will use this to accurately put the TTC solution and yeast suspension in to the test tubes. I have decided to use a syringe over another piece of equipment such as a measuring cylinder because a syringe is more accurate and precise because a measuring cylinder is to big to see if the volume that is being taken is the exact amount where as a syringe can easily be adjusted to acquired the correct volume of TTC or yeast.
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Distilled Water (5cm3): -
I will need this to dilute the TTC-Yeast mixture for the colorimeter.
Reasons for why I rejected the other methods: -
I had a choice of three other experiments that I could use co carry out this test: -
1. Hydrogen Carbonate indicator
2. Redox indicator (Methylene Blue with oil)
3. Gas Burette
4. Monometer Tube
1. Hydrogen Carbonate indicator
This method shows a colour change of the hydrogen carbonate indicator from red to yellow. This is done when carbon dioxide is added to the indicator in this case from the waste product of respiration. This experiment works similarly to the method using TTC except this indicator uses the carbon dioxide to change colour. As the temperature increases then the time taken for the hydrogen carbonate indicator to change colour will decrease and the rate of reaction will increase. This method has a few advantages such as the hydrogen carbonate indicator can be placed in to a colorimeter to find the end point with out having to dilute the indicator because it is not thick. The solution can also be placed in to the colorimeter easily because once it has changed to the colour u want it to and remove it from the delivery tube it will not change colour any further as there is no carbon dioxide being added to the solution where as using the TTC or methylene blue experiment you have to add an inhibitor which will make the experiment inaccurate because by the time you would add the inhibitor the TTC solution would have further changed colour. The use of a colorimeter in this experiment would make the results reliable. Although this experiment has its advantages it also has disadvantages, limitations and large sources of error. The reason that I didn’t choose this method is because it has a lot of sources of error such as judging the colour change by eye making the experiment inaccurate although this is the same with TTC there is another disadvantage which is that it takes longer because there is less carbon dioxide coming out of the metabolic pathway therefore if the yeast is not that active the colour change will either take very long or there will not be enough carbon dioxide to change there colour. This experiment is not very accurate and reliable because the colour change has to be recorded by memory.
2. Redox indicator (Methylene Blue)
This method also involves a colour change of the methylene blue from blue to colourless. Methylene blue is very sensitive to oxygen and if in presence of it the experiment won’t work because the oxygen will stop the blue colour changing to colourless. To prevent oxygen entering the experiment a small amount of oil is added on top of the methylene blue-yeast mixture. This colour change can only occur without oxygen therefore it will work only anaerobic conditions. The methylene blue solution then turns colourless when NAD+H+ are produced. I did not choose this method because it is very unreliable as it has a lot of sources of error such as judging the colour change by eye. Even though I have to judge the colour change by eye in the Hydrogen carbonate indicator and the TTC experiments this is harder because now I am trying to spot a change from a coloured solution to colourless, which is harder to find. This experiment is also very slow, as I have to wait for the experiment to go under anaerobic conditions. This experiment does have its advantages for example it can be put in a colorimeter but an inhibitor has to be added and the whole mixture has to be diluted to find the end point of the experiment. This experiment also is not very accurate and reliable as the colour change has to be recorded by memory.
3. Gas Burette
This experiment also requires the use of carbon dioxide. When the yeast respires carbon dioxide is released from the metabolic pathway because the Decarboxylase enzyme removes it. When the carbon dioxide is released it passes through the delivery tube and in to the gas burette, which is full of water. As the carbon dioxide is passed in to the gas burette the water is displaced down, a reading of how much carbon dioxide released can be found by looking at how much water has left the gas burette. This experiment has an advantage over the TTC and Methylene blue experiments because a judgement of the eye is not required only on reading the gas burette which is not as bad as trying to keep the colour of the experiment the same through out the whole experiment by using your memory. I did not choose this experiment because it has a very small chance of working because if the yeast is not very active then very little if any water will be displaced and if the yeast is very active or at a high temperature the water will be displaced to quickly as a fixed time is set for the experiment so it will be hard to read the end reading as water is continuously being displaced. This experiment is not reliable because by the time you try to read the final meniscus level more water has been displaced also making the experiment inaccurate.
4. Monometer Tube
This experiment also involves the use of carbon dioxide. This time when the yeast respires and releases carbon dioxide it passed through the delivery tube and in to the monometer tube. The carbon dioxide the give pressure on to the coloured liquid inside the tube and forces it to move. A curtain time is set for the experiment for example 30 seconds and the movement of the coloured liquid determines the rate of respiration. This experiment also has an advantage to the fact that there is not a big source of human error by using the eye and memory. I did not choose this method because the monometer tube is very sensitive to carbon dioxide therefore if the yeast is not very active or the experiment is at a low temperature then you will still get a good result, although this seems like an advantage its not because if the yeast is very active or the experiment us at a high temperature then the reaction will work to fast and the coloured liquid will move more quickly making it hard to read the end point.
Reasons for choice of the method that I am using: -
The reason I chose the method that I chose is because it is quick, easy reliable to the extent that the colour change colourless t pink is easy to spot. Another advantage for this experiment is because even if the yeast is not active then a colour change will still occur because of the amount of hydrogen being removed from the metabolic pathway is more than carbon dioxide so this experiment has an advantage over the other methods. The end product of this experiment can also be added to a colorimeter to find the end point and make the results more accurate and reliable.