Pyruvate [C3]
(The stages of glycolysis with regards to our experiment)
Overall, per glucose molecule, glycolysis produces;
- 2 molecules of ATP (4 are produced but 2 are used up in glycolysis)
- 2 molecules of reduced NAD, which later feeds electrons into the electron transport chain.
- 2 molecules of pyruvate, which enters the ‘link’ reaction of oxygen is available.
In the presence of oxygen, yeast is able to go through aerobic respiration in which the pyruvate formed in glycolysis passes into the matrix of the mitochondria and the sequence of events called the ‘Kreb’s cycle’ occurs.
We can summarise the overall reaction of the ‘Kreb’s cycle’ as follows;
- Two carbon dioxide molecules enter the cycle and two carbon atoms are lost as carbon dioxide.
- One molecule of ATP is synthesised.
-
Four pairs of hydrogen atoms are removed, three NAD+ molecules are reduced to NADH and one FAD molecule is reduced to FADH.
- The Krebs cycle turns twice per glucose molecule.
In the absence of oxygen, yeast must respire anaerobically, since without oxygen, the electron transport chain is not able to function properly. Instead, reduced electron carriers accumulate in the cell. This would normally be a problem because eventually, all of the NAD+ would be converted to NADH and all of the FAD to FADH2 and metabolism would stop.
Yeast cells are able to overcome this problem and reoxydise the reduced electron carriers by converting pyruvate into acetaldehyde using a decarboxylase enzyme, which removes carbon dioxide from pyruvate. Acetaldehyde is then reduced by NADH to give ethanol and NAD+ .
Glucose (sucrose is glucose and fructose)
2ADP + Pi NAD+
2ATP NADH + H+
NADH + H+ NAD+
Pyruvate Acetaldehyde Ethanol
Carbon Dioxide
(Source- Nelson advanced science; Respiration and Coordination)
Other factors affecting Enzymes:
- Substrate concentration- at very low substrate concentrations, collisions between enzyme and substrate molecules are very infrequent the reaction proceeds slowly. This is because in low concentrations there is a low quantity of substrate molecules to collide with the active sites of the enzyme. The rate of reaction will increase as the substrate concentration increases, but only until all the enzyme molecules are being used. After this point, no matter how much more substrate is added, the enzymes are working as fast as they can so the rate reaches a maximum velocity and stays constant. This rate is referred to as V max.
- Enzyme concentration- as the concentration of enzymes increases, so does the number of active sites. Therefore the rate of reaction increases in proportion to the concentration of the enzyme.
- pH- enzymes have a characteristic pH at which they function most efficiently. The name for this pH is the optimum pH. Changes in pH affect the shape of the enzyme’s amino acid residues. This affects the overall shape of the enzyme’s molecule and therefore affects the efficiency of formation of enzyme substrate complexes. Really high or really low pHs may even denature the enzyme.
- Inhibitors- competitive inhibitors are substances that compete with substrates to get to the active site. If an inhibitor goes in place of the active site, it blocks off all chances of substrates binding to it, and therefore slows the metabolic rate.
- Non competitive inhibitors join to a place on the enzyme known as the allosteric site. When it does this, it alters the enzymes bonds and changes the active site, meaning the enzyme cannot perform and is said to be denatured. This will drastically slow down the metabolic rate.
(Source- Nelson Advanced Science; Molecules and cells).
Equipment list:
- Pipettes (1cm³ and 3cm³) - to measure small amounts of substances in order to make measurements more accurate.
- Water baths × 5 – these ensure that all of the boiling tubes are kept at a constant temperature and do not lose or gain heat.
- Boiling tubes × 5 – for testing the yeast balls in.
- Clamp and clamp stand – to make sure that the yeast balls are all dropped from the same height to ensure that they all have the same spherical shape.
- Filter paper – For filtering any excess calcium chloride from the immobilised yeast balls.
- Funnel – to guide the excess calcium chloride which has been filtered from the yeast balls into a beaker.
- Measuring cylinder × 2 – To allow for accurate measurements of substances. One needs to be used for each type of substance to avoid contamination.
- Syringe- to measure out substances and to allow for the yeast mixture to be dropped into the beaker containing the calcium chloride solution.
- Test tube rack – to allow for the boiling tubes to be held in a steady position.
- Thermometer- for measuring the temperature of the boiling tubes whilst in their water baths.
- Glass rod- for the agitation of the yeast mixture
-
Beakers × 3- separate beakers are needed. One is used to make the yeast mixture; the other is used to hold the calcium chloride solution into which the mixture will fall. A beaker must also be used to mix the sucrose solution and water to make standard dilutions.
- Stop watch- in order to time how long it takes for the beads to rise to the surface.
- Electronic weighing scale- to measure yeast for increased accuracy.
Chemicals used;
- 1.5% Calcium chloride solution
- 4% sodium alginate
- Sucrose (0.5M, 1M, 1.5M and 2M)
- Distilled water
- Saccharomyces cerevisiae (baking yeast)
Method:
-
Firstly, the yeast needs to be immobilised. In order to do this we must weigh out 3 grams of saccharomyces cerevisiae (fresh baking yeast) using the electronic weighing scale. Then measure out 5cm³ of distilled water using a measuring cylinder and mix it with the yeast thoroughly using a glass rod until all the yeast is dissolved. When measuring out 5cm³ of water, take care to ensure for accuracy by checking the measuring cylinder at eye level and by topping it off using a pipette if necessary.
- Using a suitably sized syringe, measure out 15cm³ of sodium alginate, then add it to the mixture and mix it together using a glass rod, ensuring that it is all at the same consistency. Sodium alginate is used due to it being a gel which allows the yeast to be contained in little balls.
- When the mixture has been thoroughly mixed, we must pour it into a syringe, which is held in place using a clamp. A clamp must be used in order to make the yeast balls the same size and consistency.
- Measure out 50cm³ calcium chloride solution and pour it into a beaker. We must then put the calcium chloride solution under the syringe and allow for the yeast mixture to drop into the beaker and form little beads of insoluble calcium alginate, containing the trapped enzyme. The height of the syringe needs to be tested and adjusted to ensure that each bead is perfectly spherical. Whilst the mixture is dropping into the calcium carbonate solution, the beaker must be swirled and kept moving to ensure that the beads remain separated.
- Once all of the mixture has been poured into the beaker containing the calcium chloride solution, the beads must be left in the beaker to set for 15 minutes to harden.
- Once left to set, using a funnel and filter paper, the beads can be filtered and then washed using tap water.
Preliminary experiment:
Aim:
Find out which concentration of sucrose solution is best to allow for the yeast beads to rise.
Method:
-
To make standard dilutions, using a measuring cylinder and pipettes, accurately mix part 1M concentration of sucrose solution with part distilled water, ensuring that the total volume is equal to 20cm³ each time to make a new concentration of sucrose.
- Add the new solutions to a boiling tube and place the boiling tubes into a test tube rack.
- Place 3 immobilised yeast balls into each new concentration and time how long it takes for them to rise to the surface. All preliminary experiments are done at room temperature.
- Record all results in a table in order to choose a suitable concentration to use for the real experiment.
Results table
Discussion of preliminary results:
Yeast beads were rising up to the surface far too quickly in all the given concentrations of sucrose (2M, 1.5M, 1M and 0.5M). In these cases, beads rose to the surface of the boiling tubes in a second. This is largely due to the buoyancy of the solutions, rather than because of the respiration of yeast forming carbon dioxide. For this reason, these concentrations would not be suitable for the actual experiment because of their density.
Since the given concentrations were too dense, a suitable concentration was obviously between 0M and 0.5M. When testing the 0.35M concentration of sucrose, the mean time taken for the beads to rise was 153.73 seconds. I found that this was still quite fast; however, the time taken was long enough to prove that the yeast was respiring and that the beads were not floating due to the buoyancy of the sucrose. Although this would be a suitable concentration to use for the actual experiment, the three different results that I acquired from this test were too spread out. I therefore didn’t think that this concentration would be reliable.
I then tested 0.25M concentration, which produced a mean time of 486.97 seconds. This was a good time due to it being quite slow, but I hoped that at different temperatures it would be quick enough for me to use in the actual experiment.
When testing a 0.15M solution, a mean time of 902.13 seconds was produced. I felt that this time produced took too long and would not be very good in my actual experiment.
For my actual experiment, testing the effect of temperature on the respiration of yeast I decided to use a concentration of 0.25 because it was easy to produce and allowed for accurate results. This is handy since it allows me to put more beads into a test tube and test the time for each of their rising to the top, giving me a larger sample size and therefore a more accurate range of results.
Experiment to find out the affect of temperature on the respiration of yeast:
Method:
-
Accurately measure out 25cm³ of 1M sucrose solution and 75cm³ of distilled water using a measuring cylinder (top off with a pipette to ensure accuracy) and pour them both into a beaker to make a 0.25M solution.
-
Pour 20cm³ of the new 0.25M solution into 5 boiling tubes. Take care to avoid spillages.
- Place one boiling tube into one of the water baths. Put a thermometer into the boiling tubes and wait for the solutions to warm up to the correct temperature. When the solutions have reached the correct temperature, place 5 immobilised yeast beads into the boiling tube and using the stopwatch, time how long it takes for each bead to rise to the surface. When all times have been gathered, record results in a results table.
-
Repeat this procedure for 33°C, 42°C, 48°C, 54°C, 62°C and 70°C.
- When 1 set of results have been gathered rinse off all boiling tubes, make more standard dilations of 0.25M sucrose solution and repeat the whole experiment again twice.
A volume of 20cm³ of sucrose solution was used since it allows for a suitable distance for the immobilised yeast beads to rise to the surface. This distance allowed me to have enough time to get ready with the timer and record results.
I used 5 immobilised yeast balls at one time because I felt that it was manageable and that they would not produce too much waste ethanol to poison the enzymes and cause slower reaction times.
Fair testing:
There are many factors which affect the rate of respiration. However, to maintain a fair test, I must make sure that I only change one variable (in this case temperature). To do this I must…
- Use the same concentration of sucrose each time. In the case of my experiment, use 0.25M. When making the 0.25M standard dilution I must take care for accuracy when measuring out the sucrose solution and water to avoid the wrong concentration being made.
- Make sure that the enzyme concentration remains constant by making equal sized beads from one thoroughly mixed mixture which is all at the same consistency. Another reason why the beads need to be the same size and shape is to ensure that they all have the same surface area. To do this, I must use a clamp to make sure the height at which the mixture drops at is the same at all times.
- pH should be kept constant at all times. A buffer solution however is not necessary due to all of the solutions being the same pH.
- Although temperature is the variable I am changing, boiling tubes need to be kept in a water bath to ensure that no heat is gained or lost when conducting my experiments. A thermometer needs to be kept in the water bath to prevent this.
- Avoid contaminations from substances such as vitamin C and ethanol which are also in the lab, as these will act as inhibitors to the enzyme.
Safety:
When carrying out this experiment, great care needs to be taken when handling equipment, due to them being made out of glass. I must ensure to keep boiling tubes in a test tube rack to stop them from rolling off tables. I must also wear safety goggles when handling calcium chloride which is said to be a skin and eye irritant. If any calcium chloride or any other chemicals spill on me, I must ensure to wash my hands thoroughly and to tell the member of staff supervising the experiment.
Results:
Summary Tables:
Discussion:
As the temperature of the sucrose solution increased, the time taken for the yeast beads to rise decreased until it reached a minimum point (the optimum temperature) and from there began to increase. This has been shown in my graph.
The rate of reaction showed a steady increase as the temperature increased. On my graph this is indicated between 0 and 48°C. This is because a rise in temperature causes the enzyme and substrate molecules to vibrate more. This increases the chances of enzyme-substrate complexes being formed since they have more energy to collide. In the case of this experiment, as the temperature increases it allows for pyruvate to be produced more quickly because the hexokinase enzyme has more energy and so is able to collide with substrate molecules at a faster rate. This pyruvate is then able to be converted into acetaldehyde more quickly, using a decaboxylase enzyme, which removes a molecule of carbon dioxide from the pyruvate. With higher temperatures this carbon dioxide is able to be produced at a faster rate, which makes the yeast beads rise quicker.
However my graph shows that the rate of reaction rose to a maximum point (optimum temperature) and from there started to decrease. My graph indicates that around 48ºC is the optimum temperature due to this being the point after which the graph starts to slope downwards again. This happens because the enzymes begin to vibrate to hard that their hydrogen bonds and ionic forces break apart altering the active site meaning that the hexokinase and decarboxlase enzymes cannot function, and carbon dioxide is not produced as rapidly. This means that the beads take longer to rise.
The difference in rate increases at a proportional rate of 2.36 1000/s from 0 to 33°C. However, from 33 to 42°C this difference in rate decreases considerably from 2.36 1000/s to 0.39 1000/s, which is 6 times less than the previous difference, therefore forming a levelling off effect on my graph. This does not follow the normal trend and is likely to be an anomalous result. However, I will discuss this further in my evaluation. At the maximum rate of reaction which occurs at 48°C (possible optimum temperature) the rate has increased by 1.10 1000/s. At 54 °C the rate begins to decrease to 2.63 1000/s. This is a similar result to the rate at 48°C, forming a parabola shaped graph. From 54ºC onwards the rate begins to decrease steadily. This is evidence that temperature has had an effect on the respiration of yeast meaning that I can disregard my null hypothesis.
To measure the spread and accuracy of my results, I can use the formula….
(∑x² - [(∑x)²/n])
n.
This formula allows me to calculate the standard deviations of all the data with which I can mark on my graph using error bars. These error bars will be talked about in greater detail in my evaluation.
Standard deviations:
Graph 1: Time taken under increasing temperatures (seconds)
-
38°C
-
√(419.84² + 423.28² + 427.66²) – [(419.84 + 423.28 + 427.66)²/3]
3
= 3.6
-
42°C
-
√(366.32² + 353.32² + 370.8²) – [(366.32 + 353.32 + 370.8)²/3]
3
= 7.4
-
48°C
-
√(286.16² + 250.44² + 242.12²) – [(286.16+ 250.44+ 242.12)²/3]
3
= 18.7
-
54°C
-
√(380.22² + 379.62² + 379.9²) – [(380.22 + 379.62 + 379.9)²/3]
3
= 3.2
-
62°C
-
√(655.64² + 676.84² + 673.12²) – [(655.64 + 676.84 + 673.12)²/3]
3
= 11.4
-
70°C
-
√(954.72² + 960.12² + 967.06²) – [(954.72 + 960.12 + 967.06)²/3]
3
= 9.5
Graph 2: rate of respiration under increasing temperatures (1000/s)
-
38°C
-
√(2.38² + 2.36² + 2.34²) – [(2.38 + 2.36 + 2.34)²/3]
3
= 0.02
-
42°C
-
√(2.73² + 2.83² + 2.70²) – [(2.73 + 2.83 + 2.70)²/3]
3
= 0.15
-
48°C
-
√(3.49² + 3.99² + 4.13²) – [(3.49 + 3.99 + 4.13)²/3]
3
= 0.48
-
54°C
-
√(2.63² + 2.63² + 2.63²) – [(2.63 + 2.63 + 2.63)²/3]
3
= 0
-
62°C
-
√(1.53² + 1.48² + 1.49²) – [(1.53 + 1.48 + 1.49)²/3]
3
= 0.02
-
70°C
-
√(1.05² + 1.04² + 1.03²) – [(1.05 + 1.04 + 1.03)²/3]
3
= 0.0082
Quartiles and the interquartile range;
In order to find any more anomalies in my results, it is necessary for me to find the interquartile range (the third quartile [Q3] minus the first quartile [Q1]).
The formula for finding outliers is 1.5(Q3 - Q1) and in order for me to find the quartiles, I must first put all of my results in order starting with the smallest first for each temperature individually. All outliers can then be disregarded to give a graph more suited to the expected graph.
Number of results (n) for each temperature= 15
Q1 = ¼ n = 3.75 (4 rounded up)
Q3 = ¾ n = 11.25 (12 rounded up)
-
38°C
412.3, 413.5, 414.6, 416.3, 418.9, 419.5, 421.3, 422.5, 426.9, 428.6, 430.2, 430.2, 431.4, 431.5, 436.2
4th value = 416.3
12th value = 430.2
1.5(430.2 – 416.3) = 20.85
Since there are no 2 consecutive results in the arranged order which have a 20.85 or above difference, there are no outliers.
-
42°C
307.8, 310.2, 314.5, 323.2, 325.6, 328.2, 351.8, 356.2, 389.2, 390.9, 391.9, 406.1, 407.6, 423.1, 425.9
4th value = 323.2
12th value = 406.1
1.5(406.1 – 323.2) = 124.35
Since there are no 2 consecutive results in the arranged order which have a 124.35 or above difference, there are no outliers.
-
48°C
186.8, 199.2, 200.3, 220.5, 246.2, 248.3, 250.6, 255.4, 258.9, 273.7, 283.8, 288.1, 295.3, 390.0, 396.5
4th value = 220.5
12th value = 288.1
1.5(288.1 – 220.5) = 101.4
Since there are no 2 consecutive results in the arranged order which have a 101.4 or above difference, there are no outliers.
-
54°C
345.5, 360.1, 360.2, 361.8, 362.5, 363.3, 363.7, 370.2, 378.2, 380.3, 380.3, 410.7, 413.2, 417.3, 431.4
4th value = 361.8
12th value = 410.7
1.5(410.7 – 361.8) = 73.35
Since there are no 2 consecutive results in the arranged order which have a 73.35 or above difference, there are no outliers.
-
62°C
640.2, 652.1, 655.1, 658.2, 660.5, 662.3, 664.3, 670.2, 670.3, 672.1, 675.1, 677.8, 680.5, 690.2, 699.1
4th value = 658.2
12th value = 677.8
1.5(677.8 – 658.2) = 29.4
Since there are no 2 consecutive results in the arranged order which have a 29.4 or above difference, there are no outliers.
-
70°C
921.3, 923.2, 934.6, 936.1, 936.5, 937.5, 938.2, 954.3, 961.3, 982.1, 992.7, 995.3, 996.7, 997.1, 1002.6
4th value = 936.1
12th value = 995.3
1.5(995.3 – 936.1) = 88.8
Since there are no 2 consecutive results in the arranged order which have a 88.8 or above difference, there are no outliers.
Due to there being no outliers in my results, I can say that all my results follow a pattern to show the characteristics of enzymes.
Evaluation:
The results that I gathered in this experiment support my hypothesis; since I predicted that the rate of reaction would increase with increasing temperature until reaching the maximum point (the optimum temperature) after which it begins to decrease again.
When carrying out my actual experiment, to improve accuracy and reliability I put 5 yeast beads into the boiling tube at once and repeated this 3 times for every temperature. This means that I would have 15 separate results for each different temperature, allowing me to calculate mean results. Since so many results were taken I can be fairly sure that results were reliable and accurate. Since my graph reflects the predicted graph shown in my prediction, this reinforces the reliability of my findings.
When discussing results in my analysis, I discussed the fact that because of a levelling off effect at 42°C that this was a possible anomalous result. However, when carrying out statistical testing, I found that because the highest standard deviation products occurred at the 48°C result for both the rate of reaction and the time taken for the reaction graphs (indicated using error bars on my graph), the levelling off affect may have been caused due to inaccuracies at 48°C.
There are many ways in which these accuracies could have occurred in my actual experiment. One of these was that since yeast beads had a tendency to rise up to the surface at similar times, it was difficult to record the times of 2 or more beads that were coming rapidly up to the surface. This may have caused minor inaccuracies in my results. To prevent this type of inaccuracy happening again in the future, a split timer could have been used which records the time of more than one beads at a time without having the need to restart the timer. Another problem that we faced while carrying out the experiment was that due to the boiling tubes all being inside a water bath, it was extremely difficult to see parts of the boiling tube due to the refraction that occurs in the water. This meant when we thought the beads had reached the surface of the sucrose solution inside the boiling tubes; the possibility occurred that this could have just been an optical illusion causing more inaccuracies in our results.
Another reason for inaccuracies could have been that fact that the immobilised yeast beads weren’t all the same size. Although the height from which they were dropped was controlled by a clamp and clamp stand, many of the beads could have landed awkwardly into the calcium chloride solution, thus causing some beads to have differences in diameter. However, beads were chose at random to prevent their size being too much of an issue. To prevent this type of problem happening in the future, a sieve could be used to filter out beads that are slightly bigger than the rest.
Although I thought that 5 beads was a good number to have in each boiling tube when conducting each experiment, due to ethanol being a waste product of anaerobic respiration, it is possible that since ethanol is a substance that acts as a non competitive inhibitor to hexokinase and decarboxylase enzymes, the release of this product from one bead could have affected the metabolic rates of the other beads and therefore could have affected their results. If there was more time to conduct this experiment it would have been beneficial to use 5 test tubes for each temperature with 1 yeast bead in each test tube.
If I had the chance to conduct this experiment again, there are a number of factors that I would change to make the overall experiment more accurate and therefore more reliable. One factor that I would change is the fact that instead of using a syringe to measure out volumes, I could have used a burette. This is a more effective way of measuring out volumes due to the fact that fewer inaccuracies would occur. A burette would also be a suitable method of expelling the yeast/alginate mixture, since every drop that is expelled is of the same volume. Another way in which this experiment could have been improved is by giving us more time to conduct the experiments. This would mean that we can repeat experiments a greater number of times, while using less beads in each boiling tube to reduce the risk of the yeast beads being poisoned and thus denatured by their own waste.
Overall, the completion of my experiment was a success as indicated by the statistical test which I carried out, involving finding the quartiles to allow me to work out if there were any outliers or extreme results. Since there were no extreme results found, I can assume that the results that I acquired from my experiment were quite accurate and therefore followed a suitable pattern to explain the characteristics of enzymes.
To extend this investigation, there are a number of different variables that we could test. For example, substrate concentration, pH, inhibitor, enzyme concentration etc. In order to extend this inquiry I have produced a short investigation on how inhibitors affect the rate of reaction.
Hypothesis:
As the concentration of ethanol increases, the longer it will take for the yeast beads to rise to the surface.
Method:
- Put a test tube rack with 5 boiling tubes into a 50°C water bath to ensure that all experiments are carried out at the same temperature in order to keep this a fair test.
- Make standard dilutions of 0.25M of sucrose solution by measuring 75cm³ of distilled water and mixing it with 25cm³ of 1M sucrose. Then divide this into 5 and pour it into the 5 boiling tubes.
- Make standard dilutions of ethanol concentrations, corresponding to the following table
- Add a different concentration into each of the boiling tubes and continue to place 5 yeast beads into the solution. Time using a stopwatch how long it takes for each bead to rise to the surface and record results in a results table.
- Repeat these experiments 3 times in order to work out a mean time for each experiment.
Sandeep Tailor 12.6
Biology Coursework 2004.
Centre number; 12416
Candidate number; 8534