- compact
- insoluble
- readily accessible when needed
When treated with an iodine/potassium iodine solution, starch gives a characteristic blue-black colour (which is starch-iodine complex). This reaction has been used in the experiment to demonstrate and detect the presence and amount of starch produced.
The biochemical reaction in which starch is produced is shown below.
We can interpret the illustration as following: glucose needs both enzyme (glucose pyrophosphorylase) and ATP to form glucose-1-phoaphate, which is in turn activated by ATP and another enzyme (ADP- glucose pyrophosphorylase) to form ADP glucose. The enzyme starch synthase catalyses the addition of glucose monomers onto the end of the growing starch chain.
It is clear that both ATP and enzymes play an important role in the starch synthesis.
Hypothesis
Due to the fact that enzymes are sensitive to temperature and PH changes, and that they are crucial to the synthesis of many chemicals, we can demonstrate how enzymes are affected by temperature and PH changes by detecting the presence and amount of products which would not be produced without enzymes.
Prediction
- More starch is likely to be produced at 30 C than at room temperature, because 30 C is a more desirable temperature for enzymes to work (most enzymes have an optimum temperature around 30 C to 40 C, at which they can work most effectively).
- The longer the reaction between enzymes and substrates takes place, the deeper the colour after terminating the reaction because more starch is produced (since ADP-glucose can be produced to add onto the end of the starch chain to elongate it).
- If the potato extract used contains all these enzymes-- glucose pyrophosphorylase, ADP glucose pyrophosphorylase and starch synthase, glucose can produce starch but most slowly since it undergoes the process of forming glucose-1-phosphate first. So the amount of starch produced in the same time interval will be: experiment 2 (glucose at 30 C) < experiment 3 (glucose-1-phosphate at room temperature) < experiment 1 (glucose-1-phosphate at 30 C).
- If the potato extract used does not contain glucose pyrophosphorylase which catalyses glucose to form glucose-1-phosphate, there will be no starch produced in experiment 2.
Method
Three experiments will be carried out:
- with glucose-1-phosphate at 30 C
- with glucose at 30 C
- with glucose-a-phosphate at room temperature
- Label 8 test tubes to receive 3 cm of 1% glucose-1-phosphate
- Label 4 test tubes to receive 3 cm of 1% glucose
- Label 12 test tubes to receive 5 cm of standard iodine solution
- Label 12 miniature tubes to receive 1 cm of potato extract
- When ready to begin the experiment, add 1 tube of potato extract to each tube of substrate. Note the time.
-
Immediately i.e. zero time tip one tube of the reaction mixture into a tube of iodine. The iodine denatures the enzyme and stains any starch present.
- At intervals of 20 minutes tip another tube of reaction mixture into iodine. Repeat for a total of 4 tubes.
- At the end of the experiment determine the amount of starch in each tube by reading the depth of colour in the colorimeter. Record the results and repeat the experiment for the other 2 treatments.
- Plot light absorption against time for each experiment.
The colorimeter consists of a light source, a filter to select the appropriate range of wavelengths, a photocell whose electrical resistance is proportional to the intensity of light falling on it, and a meter. The sample is then places between the filter and the photocell, a simple diagrammatic representation being given below.
N.B.
- Iodine solution was used here because:
- It terminate the reaction by denaturing the enzymes—the hydrogen bonds and ionic bonds that maintain the structure of enzymes are broken thus enzymes lost their function.
- It gives a characteristic blue-black colour when reacting with starch, which is often used to detect the presence and amount of starch.
- Before using the colorimeter to measure the samples in experiment one, set up the zero point by putting the sample with the least intensive colour in the filter. Repeat this process during experiment two and three.
- Shake the samples to make the starch evenly spread in the test tubes before measuring.
- The readings do not tell us how much actual starch is produced but tell us whether the amount of the starch in one sample is more or less than that in another sample by recording the intensity of light falling on them.
Variables
Independent variables: temperature at which the experiment was taking place; time intervals; the volume and concentration of substances used.
Dependant variables: change in colour
Results
Conclusion
It can be clearly see from the result that starch was produced in glucose-1-phosphate both at room temperature and at 30 C water bath whereas no starch was produced in glucose at 30 C water bath. In the two starch-yielding groups, we can also see that the longer time the reaction took, the deeper the colour, hence the more starch was produced. Finally, more starch was produced in glucose-1-phosphate at 30 C than at room temperature.
Discussion
We can see from the result that the enzymes in the potato extract catalysed glucose-1-phosphate both at room temperature and at 30 C, but did not catalyse glucose. From this observation we can demonstrate that the enzymes present in the potato extract are ADP glucose pyrophosphorylase and starch synthase. Because there was not the right kind of enzyme-- glucose pyrophosphorylase to catalyse glucose, no glucose-1-phosphate was produced in experiment 2 thus no starch was produced. The specificity of enzyme is demonstrated by this result.
Although the enzyme can catalyse glucose-1-phosphate both at room temperature and at 30 C, more starch was obtained at 30 C, which can be explained by the fact that enzymes are most active, in other words, can work most effectively at optimum temperature. For most enzymes, the optimum temperature is around 30 C to 40 C, so 30 C was the more desirable temperature for the enzymes, thus they can catalyse more substrates to produce starch than at room temperature.
Errors might have occurred in the experiment due to:
1. Although a special pipette has been used, personal inaccuracy might still have occurred when pipetting the potato extract to the miniature tubes (slightly more or less than 1 cm of potato extract might have been added into each tube), which might have led to inaccurate results. Accuracy can be improved by using a volumetric pipette.
2. One container was used for all the samples in the same experiment when using the colorimeter, so there were always a few drops from the previous sample left in the container, which might have affected the reading of the next sample. We can wash the container every time before reusing or use a new one for each sample.
3. The biggest source of error in the experiment was time controlling.
-
In step 6, I was required to immediately tip one tube of the reaction mixture into a tube if iodine. Whether I was slow of fast in this step might have affected the result.
- I used a two-handed watch in the experiment, so I was unable to keep the intervals at exactly 20 minutes. A three-handed watch or even a stopwatch would be much more accurate.
The method can be improved by interfacing the colorimeter to a computer to obtain a more accurate reading result.
A further study could repeat the experiment at different time intervals and longer time, say 10 minutes for 2 hours, to give more data on the production of starch. Also different enzymes and substrates from different types of organisms (e.g. animals and bacteria) could be employed. The effect of different temperature and chemicals (acids and bases and some inhibitors) could be investigated as well.
Bibliography
- Collins Advanced Science Biology
- Understanding Biology For Advanced Level
- Oxford Dictionary Of Biology
- Website: http://www.biocarta.com
YIN WENYI
27 November 2002
