Effect of Temperature of Starch Phosphorylase

Introduction
This practical experiment was performed in an attempt to observe how enzymes (starch phosphorylase in particular) are affected by varying its temperature before introducing it to the substrate it will be reacting with. A catalyst (enzyme) is a substance that changes the rate of a reaction; for a reaction to take place at all, the enzyme must first come into contact with the substrate. Enzymes are subject to a number of factors which effect how fast they can cause a reaction with a substrate; these factors include temperature, pH levels, chemical substances and the concentration of both the enzyme and substrate. By controlling these variables we hoped to observe specifically how temperature affected the enzyme’s rate of reaction.
Enzymes can only work optimally within a certain pH range. Altering the acidity or basicity of the pH can cause alteration in the shape of the enzyme’s active site which effects how well the substrate and enzyme fit together. Most enzymes work at an optimum pH level that is neutral or slightly acidic.
Some chemical substances can interfere with the function of an enzyme by being binded to the active site or by combining with the enzyme in such a way that the shape of the active site is denatured. Chemical substances like pesticides and drugs work by attaching themselves to the enzyme of a pest or disease and restricting their functionality.
The rate of reaction can also be affected by the amount of substrate or enzyme present and how much product has been accumulated. The higher the concentration of substrate there is, the faster the reaction will occur due to there being more active sites being used and thus increasing the amount of reactions. The more substrate you add, the reaction rate should increase proportionally until enzyme saturation is reached; enzyme saturation happens when all the active sites are being occupied in which case adding more substrate will no longer effect the reaction rate.
Raising or decreasing the temperature will increase or decrease the amount of kinetic energy that molecules possess. The change of temperature will determine the speed of interaction between substrate and the enzyme by changing how often the substrate molecules randomly collide with the catalyst molecules, and in turn the rate of reaction. Although increasing the temperature increases the amount of kinetic energy a molecule has, it also increases the amount of molecular vibration which occurs within the molecule; this puts more effort on the bonds which bind the molecule together. Breaking the bonds in an enzyme molecule will cause the active site where the reaction takes place to lose shape and become less complimentary to the substrate shape that it is reacting with. If enough bonds are broken within the catalyst, it will become denatured and cease to function. Because of this reason, rising the temperature to increase the rate of reaction can become a hindrance if the temperature is raised too high past the optimum temperature at which the enzyme functions causing the enzymes to become denatured which in turn will slow the speed of the reaction.

Potato extract contains an enzyme starch phosphorylase; this particular enzyme reacts with glucose phosphate to produce starch. By testing the glucose phosphate and starch phosphorylase solute for the presence of starch at the same intervals of time and with controlled concentration of both the enzyme and substrate but at varying temperatures, we were able to observe temperature’s effect of enzyme activity.

Temperature’s effect on enzyme activity pH’s effect on enzyme activity

Substrate’s concentration effect on enzyme activity

Hypothesis
Based on the theory that different enzymes have different optimal temperatures at which they operate, I think we will see the best reaction at the cold temperature. Because potatoes grow in the ground (where it is cold), I think that this is the temperature at which the enzyme starch phosphorylase will react most efficiently with the substrate glucose phosphate to form starch.

Apparatus * Test tube rack and two test tubes * Funnel * Filter paper * Mortar and pestle * Potato * Water * Knife to cut potato * Dropping pipette * Means of heating potato extract * Glucose Phosphate * White spotting tile * Iodine solution * Sand * Ice, along with something to hold it & test tube in it at the same time * Timer

Method 1. Cut a piece of potato about 20mm square and proceed to chop it up further into little pieces. 2. Place these little pieces of potato into a mortar along with about half a teaspoon of clean sand and grind them with the pestle until mushy. 3. Add enough water to turn the mush into a more sloppy mixture (around 7-10 mL should do) and continue to grind for a while more. 4. Filter the potato, sand and water mush through a fluted filtered paper into a clean test tube. 5. While waiting for your solute to filter, use a pipette to place drops of glucose phosphate into the dips on your spotting tile; leave at least one spot to test your potato extract for the presence of starch. 6. Rinse the pipette and draw some of your filtered potato extract, place a drop of it into a spare cavity on your spotting tile and test it with a drop of iodine for the presence of starch (if the solute goes blue, repeat steps 1-6). 7. If the extract does not show the presence of starch, place a few drops of it into your two test tubes and a few drops into your spotting tile with the glucose phosphate; when you place the potato extract drops in with the glucose phosphate, start the timer. 8. Take observations for the solutes with potato extract and glucose phosphate at intervals of 0, 1, 2, 3, 4, 5, 10 and 15 minutes. Add an extra drop or two of iodine in at 5 minute intervals because the colour tends to fade. 9. After recording results for the potato extract and glucose phosphate at room temperature, take one of your two test tubes and chill it in ice for 5 minutes. 10. After chilling the potato extract for 5 minutes take its temperature and place a few drops of it into your spotting tile with the glucose phosphate; again, when you place the drops in with the glucose phosphate, start the timer. 11. Repeat step 8. 12. After recording results for the potato extract and glucose phosphate at a chilled temperature, take one your other test tube and heat it for 5 minutes. 13. After heating potato extract for 5 minutes take its temperature and place a few drops of it in your remaining cavities which contain glucose phosphate; remember to start the timer as soon as you place the potato extract drops in with the glucose phosphate. 14. Repeat step 11.

Crushed potato

Add drops of extract to spotting tile

Glucose phosphate solution
Filter paper

Potato extract

Temperature: Cold (5.2⁰C) (5 minutes in the ice bath) 20th May 2013 | Colour when iodine solution was added to glucose phosphate solution at the beginning of the experiment. | Yellow colour, same as iodine’s regular colour. | Colour when iodine solution was added to potato extract before it was added to glucose phosphate solution. | Dark yellow/ light orange. No purple or dark colour was observed which means that there is no/ very little starch present. | Glucose phosphate solution + potato extract | 0 min | 1 min | 2 min | 3 min | 4 min | 5 min | 10 min | 15 min | | Slightly darker than the normal colour Yellow. | Still slightly darker than normal yellow colour. No change. | No change | Dark shadowy colour forming at the bottom of the cavity around the edge. | No change since 3 min observations | Shadows getting darker around the edge. Very dark purple spot around the edge also, middle remains dark yellow. | Same as 5 minute observations; dark patch is coming towards the centre of the solution | Outside ring becoming thicker and darker. Dark spot is coming towards the middle. Smaller amount of yellow in the middle; darker yellow than usual. |
Results

Cold Test - (5.2⁰C)
0min 1min 2min 3min 4min 5min 10min 15min

Results

Temperature: Room Temperature 23rd May 2013 | Colour when iodine solution was added to glucose phosphate solution at the beginning of the experiment. | Yellow colour, same as iodine’s regular colour. | Colour when iodine solution was added to potato extract before it was added to glucose phosphate solution. | Dark yellow/ light orange. No purple or dark colour was observed which means that there is no/ very little starch present. | Glucose phosphate solution + potato extract | 0 min | 1 min | 2 min | 3 min | 4 min | 5 min | 10 min | 15 min | | Dark yellow/orange. | No change | No change | No change | No change | No change | Slight dark spot appearing at the very base of the cavity. | Dark specs observed at bottom of tile. |

Room Temperature Test

0min 1min 2min 3min 4min 5min 10min 15min

Results Temperature: Hot (80⁰C) 23rd May 2013 | Colour when iodine solution was added to glucose phosphate solution at the beginning of the experiment. | Yellow colour, same as iodine’s regular colour. | Colour when iodine solution was added to potato extract before it was added to glucose phosphate solution. | Dark yellow/ light orange. No purple or dark colour was observed which means that there is no/ very little starch present. | Glucose phosphate solution + potato extract | 0 min | 1 min | 2 min | 3 min | 4 min | 5 min | 10 min | 15 min | | Dark yellow/orange. | Yellow is darker at the bottom of the cavity and the edges are becoming a clear colour. | No change. | No change. | Clear around edges still, beginning to darken more in the bottom of the cavity. | No change. | Clearness around the edge is becoming thicker and denser. | Very few dark specs noticeable at the bottom of the tile. White sediment has formed throughout the cavity. |

Hot Test - (80⁰C)
0min 1min 2min 3min 4min 5min 10min 15min

Discussion 1. To prepare the potato extract, the potato was cut into small cubes and crushed with a mortar and pestle. The crushed potato was then filtered and the filtrate was collected for the experiment.

a) Why was only the filtrate used in the experiment?
The reason only the filtrate was used in this experiment was because only the filtrate of the potato extract was devoid of starch content. The cells in the potato contain starch, by crushing the potato the starch is released from the cells; by then filtering the crushed potato, the solute that is filtered should be clear of any starch. b) What problems would have occurred if crushed potato was used without filtration?
If the crushed potato was used without filtration, the addition of iodine to the crushed potato solute would go dark blue/purple immediately. Even though the starch has been released from the potato cells, it is still present within the solute. Without the filtration of the starch we would not be able to observe how the enzyme starch phosphorylase and the substrate glucose phosphate would chemically react to form the starch and thus not be able to observe how temperature effects this reaction. 2. Whilst the experiment is taking place what does the colour change indicate when iodine is added to the mixture of glucose phosphate and potato extract?
Whilst the experiment is taking place, a chemical reaction between the glucose phosphate and the starch phosphorylase which is present in the potato extract is also taking place. Iodine is used at the beginning of the experiment to test the potato extract and glucose phosphate for the presence of starch. The colour change in the mixture of glucose phosphate and potato extract indicates the formation of starch in the mixture. Starch is the product of the chemical reaction between glucose phosphate and starch phosphorylase.

3. In Part A you were asked to test for starch in the potato extract before you continued with the experiment. Did you extract sample contain and starch? Explain why or why not.
Our potato extract did not contain any starch. The presence of starch was eliminated by the crushing and filtering of the potato. The crushing of the potato released the starch from the potato cells and the filtering removed the starch from the solute. The reason the potato extract needed to not have any starch in this experiment is because the product of the enzyme and the substrate was starch; if this experiment had been done with the presence of starch from the beginning, we would not be able to tell what starch had been produced by the chemical reaction of the substrate and enzyme and thus wouldn’t be able to observe the effect of temperature on the chemical reaction.

4. Consider two possibilities: i) Something in the potato extract has converted glucose phosphate to starch. ii) The glucose phosphate has converted something in the potato extract to starch.
Which of the two possibilities is the more likely? Explain.
The alternative that the potato extract has converted something in the glucose phosphate to starch is more likely. This possibility is more likely because the alteration in temperature of the potato extract is what had an effect on the rate of reaction. It is likely that changing the temperature of the glucose-1-phosphate would not have changed the rate of reaction.

5. Give a simple chemical explanation of how this change could come about:
This change came about by the substrate glucose-1-phosphate binding to the enzyme; starch phosphorylase’s active site. The enzyme catalysed the reaction of glucose-1-phosphate to form starch amylose.
Starch Phosphorylase + Glucose Phosphate ---> Starch amylose

6. The potato extract was heated to various temperatures as an extension to the experiment. Discuss the effect heating has had on the enzyme activity:
The change in temperature of the enzyme can affect the activity in a few different ways. Every enzyme has an optimum temperature at which it functions, any temperature below of above this optimum temperature will generally have an adverse effect on the enzyme activity and thus the rate of reaction will be slowed. By heating up and enzyme it increases the speed of interaction between the enzyme and the substrate by increasing the amount of random movement of molecules allowing the substrate molecules to come in contact with the enzyme more often. If an enzyme is heated too much it can cause a change in the shape of the enzyme’s active site making it a worse fit for the substrate and slowing down the rate at which they react, extreme heat can cause a complete denaturation of the enzyme’s active site meaning it can no longer react with substrates. The cooling down of the potato extract seemed to work best because it would be the temperature closer to the enzyme’s optimum temperature.

Conclusion
This practical experiment has been successful in proving that temperature has an effect on enzyme activity. The enzyme starch phosphorylase was tested with the substrate glucose phosphate at different temperatures and varying rates of reaction were observed. The hypothesis that starch phosphorylase would react most optimally at the cold temperature was proved and we can assume this enzyme works best within a relatively cold temperature.

Bibliography
Alevelnotes.com (2012) Factors affecting Enzyme Activity | A Level Notes. [online] Available at: http://alevelnotes.com/Factors-affecting-Enzyme-Activity/146 [Accessed: 2 Jun 2013].
Bbc.co.uk (n.d.) Untitled. [online] Available at: http://www.bbc.co.uk/schools/gcsebitesize/science/images/gcsechem_18part2.gif [Accessed: 2 Jun 2013].
Bbc.co.uk (n.d.) Untitled. [online] Available at: http://www.bbc.co.uk/schools/gcsebitesize/science/images/gcsechem_18part1.gif [Accessed: 2 Jun 2013].
En.wikipedia.org (1962) Molecular vibration - Wikipedia, the free encyclopedia. [online] Available at: https://en.wikipedia.org/wiki/Molecular_vibration [Accessed: 2 Jun 2013].
En.wikipedia.org (1930) Potato starch - Wikipedia, the free encyclopedia. [online] Available at: http://en.wikipedia.org/wiki/Potato_starch [Accessed: 2 Jun 2013].
Jstor.org (2013) JSTOR:The reversible formation of starch from glucose-1-phosphate catalysed by the potato phosphorylase. [online] Available at: http://www.jstor.org/discover/10.2307/82391?uid=3737536&uid=2&uid=4&sid=21102274310721 [Accessed: 2 Jun 2013].
Rsc.org (n.d.) Untitled. [online] Available at: http://www.rsc.org/Education/Teachers/Resources/cfb/images/07D.jpg [Accessed: 2 Jun 2013].
Spenceley, M., Weller, B., Evans, B., Ladiges, P., Mason, M., Fullerton, K., Tsilemanis, C., Mckenzie, J. and Batterham, P. (2004) Biology: A Contextual Approach. Melbourne: Heinemann, p.168-169.