But overall the result is reasonable, and accords with my hypothesis. So I will use the same range of concentrations of sodium phosphate next time.
Repeats
In the preliminary experiment, I only did the experiment once, the results are not very reliable. So I decide to do three repeats next time. Less than three repeats will not give me enough data to plot error bars, but more than three may be too much during the limited time period(since the enzyme can not maintain their activity very long after been extracted).
To make it most efficient, I will use three sets of test tubes, in each set the test tubes contain 5 different concentration sodium phosphate respectively, same buffer 5 solution and same enzyme. Then these 15 test tubes will be incubated in the same water bath for the same time. Therefore I will have 3 sets of data, and then can work out the average to make the data more reliable.
And also, the enzyme suspension in each test tube must come from the same extract of potatoes. This would make sure the enzyme suspensions in each test tube are equally same, thus eliminating the possible effect of different enzyme suspension on the experiment.
In order to repeat 3 times, more materials are needed to do so. I will then need 15 test tubes, at least 75 of 10% sodium carbonate solution, 15cm3 of phenolphthalein phosphate and about 150g potatoes.
And also, the boiling tube is too big to incubate 15 of them in the same water bath, so I will use test tubes instead.
Variables
In this experiment, the only variable I want is the different concentrations of sodium phosphate, so to show its effect on the activity on enzyme, the other factors which affect enzyme working must be controlled, thus giving the same condition for enzyme activation in each test tube.
- The concentration of enzyme solution
For enough substrates, more concentrated enzyme will result in faster reaction since there will be more enzyme molecules available to react with the substrates. To control this variable, enzyme from the same extracted solution is added to each test tube.
- Amount of enzyme solution
The reason is the same as above. So each test tube must contain exact 1cm3 enzyme solution by using the accurate 1 cm3 syringes with the accuracy to the nearest 0.1cm3
- The temperature for enzyme activation
The temperature will affect the activity of enzyme since all enzymes have their own optimum temperature, so either lower or above that will lower the rate of reaction. Therefore, in this experiment I will put all the test tubes in the same water bath at 30 to keep the temperature in each tube the same and steady.
- The pH in which the enzymes work
Since all enzymes have their own optimum pH, either below or above that will lower the activity of the enzymes. In this experiment, the enzyme has optimum pH5, so I use buffer 5 to keep the pH steady and give the optimum pH to the enzyme in each test tube.
In some case, there may not be enough substrates for reaction during the 20-minute incubated time, so in the test tube which contains relatively more substrates, more products will be produced and. To prevent this happening, I will add exact 1cm3 of phenolphthalein phosphate to each test tube.
- Time for enzyme activation
In this experiment, if given sufficient time the enzyme will break down all the substrate in all tubes since phosphate is probably a competitive inhibitor, and it will result in no color difference between any test tubes. So I must give a suitable time period for the enzymes to work and also the same time period for all the enzymes in each test tube. According to the results of the preliminary experiment, a 20-minute period is reasonable. And also after 20-minute incubation, the enzymes are immediately stopped from working by adding sodium carbonate which will not only change the color of the solution but also denatures the acid enzyme phosphatase.
- Other unpredictable factors
To control any other factors that may affect enzyme working, I set the test tube which contains 0M sodium phosphate as a control to ensure that any other small environment changes won’t have effect on the result of the experiment.
Apparatus and materials
1x Liquidizer: To get the enzyme extract from potatoes efficiently
1x stop clock: To measure a 20-minute period
1x marker pen: To label the test tubes
1x Small knife: To cut the potatoes into pieces
2x 1cm3 syringes : with accuracy to the nearest 0.1 cm3
2x centrifuge tubes : one to contain enzyme extract, one to contain the same mass of water to balance the weight in the centrifuge.
2x 5cm3 syringes: with accuracy to the nearest 0.1cm3. One is used to add enzyme suspension, the other to add PPP in each test tube.
2x 250cm3 beaker: To contain potato pieces and extract.
5x stirring rod: with each one to stir the mix solution in each set of the test tubes.
5x beakers each with an accurate 100 cm3 mark:
500cm3 pH 5 buffer
About 40g sodium phosphate To make 100 cm3 sodium phosphate buffer solution
Balance
Spatula:
Piece of muslin (approx.12cmx12cm)
Filter paper Used to filtrate the potato extract
1x big filter funnel
Colorimeter with cuvette and 550nm filter
Kettle: To heat up the water for water bath Used to make 30℃ water bath
Thermometer
1x1000ml beaker
15x test tubes with rack
Bench centrifuge: Centrifuge the potato extract
One potato ( about 150g) :The raw material to make enzyme extract:
At least 15 cm3 1%phenolphthalein phosphate: with each one of the 15 test tubes contains 1 cm3 of it
At least 75cm3 10% sodium carbonate solutions: with each one of the 15 test tubes contains 5 cm3 of it.
Tray
Safety and environmental concern
The sodium phosphate, sodium carbonate, and buffer 5 solution in this experiment has no hazard to people. But the enzyme phophatase and phenolphthalein may cause eye and skin irritation. During the experiment, lab coat and safety glasses are required. Wash thoroughly after handling.
To avoid contaminate the environment, keep the phosphatase and phenolphthalein in tightly closed container after use.
Final Method
I will first label 5 beakers 1-5 and put the corresponding mass of sodium phosphate into each of them.
Then add pH 5 buffer to about 90cm3 in each beaker and use 5 different stirring rods to stir respectively until the solid is completely dissolved. Then with a dropper add more buffer, carefully using dropper, until the solution reaches the 100cm3 mark.
I will then cut a potato on the tray into small pieces, weigh out the mass of a 250cm3 beaker and then put the potato piece in it. Then use the balance to weigh out about 150g potato. Then place all the pieces into the liquidizer and use 5 cm3 of water to make a suspension. Put the lid back and switch on the liquidizer until all the potato pieces have turned to mashed. Then I will use the big funnel and muslin to filtrate the mashed potato and collect the liquid extracted for 15-16 cm3.
Then pour the liquid into a centrifuge tube and weigh out the same mass of another centrifuge tube, which contains water. Then place the two test tubes opposite to each other in the centrifuge, put the lid back and centrifuge for bout 5 minutes. After that, pour the supernatant into a clean test tube. This is used as the enzyme suspension.
Label 15 test tubes A1-A5, B1-B5, C1-C5 and add 5 cm3 buffer from beaker 1 to tube A1, B1, C1 by using the same syringe; then using the same syringe add 5cm3 buffer from beaker 2 to tubeA2, B2, C2 and continue this same procedure step wise to beaker5.
Add 1cm3 of the 1% phenolphthalein phosphate to each tube by a 1 cm3 syringe.
Then use a kettle to make some hot water and then mix with cold water in the 1000ml beaker. Use a thermometer to stir and add any either cold or hot water until the temperature keeps at steady 30℃. Then add 1 cm3 of enzyme suspension to each tube and mix them well with 5 different stirring rods, e.g. one rod stir A1,B1,C1and the other one stir A2,B2,C2 and so on. Place them together in the 30℃ water bath and at the same time start the stop clock. During the incubation, add any hot water if temperature drops.
After being incubated for 20 minutes, put all the test tubes out of the water bath and place them back on the rack. Add 5cm3 of 10% sodium carbonate solution to each tube and mix as before.
The tubes can now be stored in a fridge until the next day if required.
Then record the color of each test tube and measure the intensity of the pink colour using a colorimeter with a 550nm filter. Record the absorbance. Present them in a table with headings.
Then calculate the average absorbance for each concentration of sodium phosphate by using the value of A1, B1 and C1 for 0M, A2,B2 and C2 for )0.05M and continue this procedure to A5, B5 and C5.
Then use the average data to pot a graph of molarities of sodium phosphate added against absorbance.
Draw any error bars if necessary to indicate the range for each point plotted.
Prediction
I predict that the phosphate is an end product inhibitor of enzyme phosphatase, it will reduce the enzyme’s activity. So the intensity of pink color in the test tubes will decrease as the concentration of sodium phosphate increases. The pattern of the graph may be a straight line since more sodium phosphate present, more enzymes will be inhibitated.
Analysis and evaluation: The end product effect on the activity of phosphatase
Result table
Main trends and patterns
The graph shows the effect of different moles of sodium phosphate on the activity of enzyme phosphatase.
The pattern of the graph is a straight line with a negative gradient and a maximum value of absorbance when the moles of sodium phosphate are zero. When the moles of sodium phosphate increased, the decreased absorbance means less phenolphthalein been produced, thus less active of the enzyme. So, I can suggest from the graph that the activity of enzyme phosphatase is inhibited by the sodium phosphate which is an end product of this enzyme-catalyzed reaction.
When no mole of sodium phosphate was added, there wasn’t any sodium phosphate in the buffer to inhibit the enzyme, so the rate of the enzyme-catalyzed reaction was at its maximum. As I increased the moles of sodium phosphate in the solution which the enzyme worked in, the rate of reaction started to decrease, with the sodium phosphate of 0 M is 1.77, for 0.05M, the absorbance is 1.65, and for 0.3M, it’s just 0.98 since more inhibitor had been added. From the graph, they decreased by a constant value (about 3.54 unit of absorbance per 0.1M sodium phosphate increased) when the moles of sodium phosphate increased. That’s to say the amount of sodium phosphate has a linear relationship with the rate of reaction and had a negative effect on it.
The hypothesis
Explanation of results related to biological knowledge
Enzyme can catalyze metabolic reactions because their specific shapes, which fit particular substrates to their active sites and form enzyme-substrate complex, thus speeding up the reactions. So the shape of an enzyme will determine whether it can function well or not. The specific shape of an enzyme is given by tertiary structure as most enzymes are globular proteins, and the bonds between R-groups maintain the shape. If these bonds are broken, the shapes of enzyme will change, consequently changing the shape of active sites and therefore substrates can no longer fit in.
There are many reasons causing the shape changes of enzymes, e.g. temperature, pH. In this case, the shape is changed by the end-product inhibitor. The end product inhibition is a type of negative feedback commonly used to control the rate of a metabolic pathway in living things. They are often non-competitive inhibitors as they change the shape of the active site by entering the other part of the enzyme. And also, the end product inhibition is reversible as the inhibitors just form a loose association with the enzyme and may become detached to the enzyme later. After they leave, the shape of active sites will revert so that the substrates can bind with them and thus enable the enzyme to function again.
In this investigation, the enzyme phosphatase breaks PPP into phenolphthalein and phosphate. As an end product, sodium phosphate will be an inhibitor to the enzyme phosphatase. In the buffer solution where enzymes worked, sodium phosphate entered a part of the phosphatase other than the active site. Their combinations with enzymes broke or distorted some bonds between adjacent R-groups which are responsible for maintaining the shapes of enzymes, therefore the shapes of active sites changed and substrates can no longer fit in. Or according to the “induced fit model “theory, I suggest the end product may affect the ability of the enzyme to adjust themselves to fit into substrates. After they combined with the enzyme, they might have formed stronger bonds than those originally existed in the enzymes, so the enzymes were less flexible to change a bit to fit the substrates. In both situation, the end product left enzymes unable to form enzyme-substrate complex, and consequently lowered down the rate of reaction.
When more sodium phosphate was added to the buffer solution, more end product entered the enzymes and thus less enzymes were still able to function, so the rate of reaction decreased further. Therefore, the absorbance decreased as their corresponding moles of sodium phosphate increased.
If I add more sodium phosphate, I believe the rate of reaction will be 0 as all the enzymes have been inhibited.
Evaluation
Variability of results
Overall all the results coincide with the expected outcome, as the absorbance decreased when moles of sodium phosphate increased. And also, the results of each set are all within +0.1 differences. The first three points of sodium phosphate moles 0M, 0.05M, 0.10M respectively, match the graph perfectly and the percentage error for 0M is about +0.2/1.77*100%=+1.1%, for 0.05 is 0%, and for 0.10M, is about +2%. From the calculation, the percentage error for the first three is rather insignificant. But for the points of 0.2M and 0.3M, the error bar is obviously larger than the previous three. Although the last two points match the trend of the graph, they do fit the straight line, and the percentage errors for the last two are 4% and 10%, much great than the previous three. These two are considered as anomalies. I think it’s because the C4 and C5 were the last two reactions to be stopped, so the absorbance would higher than expected. So, although the absorbance for C4 and C5 are within 0.1 difference with the other two, they shouldn’t be used to calculate the average value. But these only can be discovered after the error bar has been drawn and the percentage error been calculated.
Overall the results are quite reliable, and support the hypothesis. This tentative nature of the results suggests that sodium phosphate is an end product inhibitor of enzyme phosphatase, and thus slow down the reaction.
Limitations of experimental technique
1. The concentration of enzyme suspension: Although the enzymes were extracted from the same potato and the same amount of enzyme suspension added to each test tube from the same container, the concentration of enzyme suspension can vary. Because the enzyme extract is a suspension, so if the suspension has been placed still for a moment, different parts of the suspension may contain different amount of active ingredient.
2. Repeats of the experimental: To ensure that all the reactions are carried by the same enzyme, the enzyme suspension added to each test tube must from the same extract of potato. So the amount of enzyme suspension will limit the number of repeat experiments.
3. The precision of colorimeter: The smallest division on the colorimeter is 0.05, but the interspace between them is quite big compared to the range of measurement (0~2). Sometimes the pointer lay between the divisions, so it is rather hard to determine the reading. For example, the data recorded for the second set, A2, B2, C2 are all 1.65, but actually according to my estimation, they are likely 1.65+0.01. And on the graph paper, the unit can be to nearest 0.01. So, if the colorimeter reading is precise enough to 0.01, the average points may be more precise and some points may match the pattern of the graph better.
4. Reaction time in each test tube. Since I have set three sets of test tubes, there might be a difference in time for reaction in each test tube. During the experiment, I placed all the test tubes back to the rack after being incubated for 20min in the 30℃ water bath, and added sodium carbonate from A1 to A5, B1 to B5, and C1 to C5 to stop the reaction. Although all the test tubes had been taken out of the water bath at the same time, but the temperature may not drop to room temperature immediately, so when I added sodium carbonate from A1 and to A5, the reaction might still carry on in C1 to C5. This slight time difference may result in an error to C4 and C5, as they were the last two to which sodium carbonate was added to stop the reaction. Therefore their absorbance would be higher than expected.
Improved method to address anomalies
- To make sure every test tube contains the same amount of enzyme, stir the enzyme suspension thoroughly before adding it to each test tube.
- Choose a bigger potato to get more enzyme suspension so that more repeats can be carried out.
- Use a colorimeter with more precise division, e.g. to the nearest 0.01
- Have an assistant to help me add sodium carbonate to every tube at the same time, reducing the effect of different reaction time on the experiment.
Further work
. But during the practical, it may not be possible to discover the zero point. I think it’s because the end product is a non-competitive inhibitor, so it may combine with the enzyme this moment and leave it later. So, it will just be instantaneous when all the enzymes have been combined with end product, thus the zero point will be hard to discover. But on the other hand, this suggestion support that the end product sodium phosphate is a non-competitive inhibitor and will lower down the reaction catalyzed by phosphatase.
Investigation
From the pattern of the graph, I suggest that at a certain value of moles of sodium phosphate, the absorbance may become zero. So for the further work, I will extent the range of sodium carbonate concentration. The hypothesis is the rate of reaction will continuingly decrease if I add more sodium carbonate in the buffer until the rate of reaction is 0.
First I will make 7 buffer 5 solution each contains sodium carbonate with concentration 0.0, 0.10, 0.20, 0.30, 0.35, 0.40, 0.45 mol/cm respectively. Label them from A to D.
Second I will choose a big potato to extract the enzyme phosphatase in the same way of last experiment.
Then I will prepare 4 sets of test tubes each contains 7 test tubes. Label them from A1 to D7.
Then add buffer A to test tube sets A, buffer B to test tube sets B and so on. Add 1cm of the 1% phenolphthalein phosphate to each tube by a 1 cm syringe. Stir the enzyme suspension well and add 1cm of it into each test tube and mix them well. Then put all the test tubes in the same water bath of 30℃. After 20 min incubation, and assistant and I will take them out of the water bath and add sodium carbonate to each test tube at the same time to stop the reaction.
Then use a colorimeter with precise division to quantify the color change in each test tube(from colorless to pink). Calculate the average of A1 B1 C1 and A2 B2 C2 and so on. Draw these 7 values on a graph paper and also the error bar.
Deducing from the results of last experiment, I predict that the graph pattern would be a straight line with negative gradient, and between the sodium phosphate concentration 0.35 and 0.45 mol/cm, the rate of reaction will be 0.
Investigation 2
To investigate whether the result of the investigation, the end product effect is only specific to phosphatase in the potato, or has a commonness to all the enzymes, I will carry out the following two experiments.
One will be the same process as the investigation of end product effect but use the beansprouts to extract the enzyme phosphatase. I predict that the rate of reaction decreases as the concentration of sodium phosphate increases and the pattern of the graph will be the same as the potatoes’.