The Effect That Temperature Has On the Rate At Which the Enzyme (Amylase) Can Breakdown Its Substrate
The Effect that Temperature Has on the Rate at which the Enzyme (Amylase) can Breakdown its Substrate
Aim:
The aim of this experiment is to find out the effect that the temperature has on the rate at which the enzyme (Amylase) can breakdown its substrate, which is Starch.
Amylase:
Enzyme having physiological, commercial, and historical significance, also called diastase. It is found in both plants and animals. Amylase hydrolyzes starch, glycogen, and dextrin to form in all three instances glucose, maltose, and the limit-dextrins.
Prediction:
I predict that as the temperature increases, the speed of the reaction will increase. Since temperature is a measure of the motion of particles, increasing the temperature will cause the particles to move faster. When particles move faster, more collisions occur and the collisions are more violent. This should increase the reaction rate. This is backed up by the collision theory, according to this theory; reacting molecules must collide with sufficient energy if they are to form products. (1,2)
When a particular temperature is reached, I believe the rate of reaction will dramatically decrease. I believe this because most chemical reaction happens faster when the temperature is higher. At higher temperatures molecules mover around faster, which makes it easier for them to react together? Usually, rises of 10 degrees Celsius will double the rate of reaction. This is true for enzymes up to about 40 degrees Celsius. However at 40oC the enzyme begins to be damaged, so the reaction slows. This is because the enzymes shape changes due to high temperatures (denaturation) and cannot catalyze the reaction as the shape of the active site has become imprecise therefore it can no longer combine with the substrate. By 60 degrees Celsius, the enzyme is completely denatured.
(7)
Method for calibration curve:
Prepare all the items that are listed above then put on a lab coat and wear safety goggles at all times. Switch on the colorimeter to warm it up and make sure the red filter is in place. Heat some water using the water bath until it reaches the highest temperature and work downward with the temperature.
Next, I would then proceed by carrying out a serial dilution to make starch solutions with a range of concentrations.
* I will take ten boiling tubes and label them 1-10
* In tube 1 I will measure centimeters cubed of 0.5% starch solution provided
* In tube 2 I will measure 10 centimeters cubed of 0.5% starch solution and then add 10 centimeters cubed and mixed
* In tube 3 I will measure 10 centimeters cubed from tube 2 and mix with 10centimeters cubed of water
* In tube 4 I will measure 10 centimeters cubed of solution from tube 3 and mix with 10 centimeters cubed of water
* Finally, I ...
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* I will take ten boiling tubes and label them 1-10
* In tube 1 I will measure centimeters cubed of 0.5% starch solution provided
* In tube 2 I will measure 10 centimeters cubed of 0.5% starch solution and then add 10 centimeters cubed and mixed
* In tube 3 I will measure 10 centimeters cubed from tube 2 and mix with 10centimeters cubed of water
* In tube 4 I will measure 10 centimeters cubed of solution from tube 3 and mix with 10 centimeters cubed of water
* Finally, I will continue my serial dilution then discard 10 centimeters cubed from tube ten. Which should leave me with 10 centimeters cubed in each tube.
Next, I will make up 10 colorimeter tubes of dilute iodine and shake vigorously, after which I will place in the colorimeter, then adjust the absorbance by pressing the "R" button.
After this, I will add 1 centimeter cubed from tube ten to the colorimeter tube containing dilute iodine and then shake. I will then place this tube in the colorimeter and record the absorbance by pressing the "T".
All this would be repeated for the different temperatures.
List of equipment:
Dilute iodine solution (Concentration - 0.005mol dm -3)- This will be use to detect starch
A source of 0.5% starch solution - This is the substrate used
A source of amylase solution (Different concentration) - This is the enzyme used
7 test tubes - These will be used to hold the mixture while in water bath
Measuring syringes (1cm3 and 10cm3) - Used to accurately measure the substances
Electronic water bath - At a maintained temperature, this will be used to heat the mixture
Stopwatch- This will be used to time the reaction
Colorimeter - This will be used to measure the absorbance of the mixture
Thermometer - This is used to measure temperature to the nearest oC
Glass rod - This is used to stir the mixture
Test tube rack - This is to hold the test tubes during the experiment
Justification for the choice of equipment:
I have chosen to use most of the equipment above due to accuracy and in order to prevent a high percentage error
I have chosen to use measuring syringes to measure the volumes of substances used since it is more accurate than a pipette. I will use an electronic water bath for maintaining the mixture at a temperature above room temperature since the temperature is more accurate than a water bath above a Bunsen burner. I will have to use ice from the freezer to reduce the temperature of the mixture to 10 degrees Celsius. A 100 degrees Celsius thermometer will provide temperature results of a sufficient accuracy (to 1OoC). The pH buffer range will be pre-prepared therefore I do not have to concern myself with measuring and maintaining pH levels. Test tubes are used instead of a conical flask because of its small surface area.
Variables:
Dependent variables
* Rate of breakdown of starch
* Time
Independent Variable
* Temperature of solution
Fixed Variables
* Volume of starch solution
* Volume of amylase solution
* Concentration of amylase solution
* Concentration of starch solution
* Volume of iodine
* Agitation
* Buffer
Number of observations:
I have chosen to repeat the experiment 3 times because it therefore allows me to calculate an average time. This will ensure that there are no abnormal results and it will increase accuracy. I have decided to start the temperature at 10oC and increase by 10oC each time since it will allow me to see the increase and decrease of the enzyme activity. It should also be accurate enough for me to predict an optimum operating temperature to an accuracy of 5OoC.
Method
* Put 10 cm3 of iodine into seven test tubes and places these in the test tube rack
* 30 cm3 of 0.5% starch solution are to be placed in a boiling tube and 10 cm3 of amylase solution into a second boiling tube. Then put these boiling tubes into a test tube rack and place them in the water bath at 20oC.
* Use the thermometer to check whether the water bath is at the stated temperature. Make sure you don't forget to check the temperature of each solution so that they are both the same as the temperature of the water bath.
* Mixing the contents of the two tubes with the glass rod while simultaneously starting the stopwatch starts the digestion.
* Immediately take 1cm3 of the mixture and place it in one of the dilute iodine solutions, bung this test tube and shake. Place a pure dilute iodine solution into the colorimeter (See method for calibration curve on how to use this piece of equipment) to zero it then put in the test tube with the starch and amylase in it and record the % absorbance in a table
* Repeat the step on bullet point 5 every two minutes
* Once the tenth minute result has been recorded repeat the entire experiment at the same temperature and then calculate the averages of results.
* Use your calibration curve to calculate the % of starch concentrations and add this to the table.
* Plot a graph of starch concentration (%) against time (minutes). Then work out the initial rate of reaction and record this in an appropriate table.
Carry out this entire procedure at the various temperatures at 10,20,30,40, 50 & 60oC
How I am going to calculate the rate of reaction:
At each temperature, I am to measure the rate of the enzyme-catalyzed reaction
Starch + water - sugars
I am going to measure how much starch disappears per minute at a constant temperature.
Fair test:
To ensure that the test is fair I will vary only one factor (Temperature). All the quantities will have to be carefully measured since small variations in the amount of enzyme used can make significant variations in the results. In the section where temperature is investigated, 2cm3 of water is added. This is because the starch solution is neutral and in the previous investigation (for pH) 2cm3 of the appropriate buffer was added. If the 2cm3 of water were not added then it would not be a fair test since the volumes used in each part of the investigation would be different. If they were different then this would affect the results since the solution would be of different concentrations and therefore one would react faster than the other would.
Safety Precautions:
* I will have to be careful when using the enzyme solution, as it is dangerous if it enters ones eye, so I will attempt to overcome this problem by wearing goggles.
* Iodine solution is a bleaching agent, so I will be wearing a lab coat so it doesn't bleach my clothes.
* Its is also lethal when inhaled, swallowed and ones eyes, so I'll try avoid these.
* I will also use a peg to retrieve the test tubes from the boiling water baths, if my skin comes in contact with the water from the water bath, I could suffer severe burns.
* I also will be using glassware during the experiment so I will have to be cautious about that.
* Also because the calorimeter and the water bath are electronic, I will avoid spilling any of the solutions near the electrical sockets in order to avoid an electric shock. (4,6 & 8)
Bibliography:
. Bennett, T. P., and Frieden, E (1969) Modern Topics in Biochemistry, pg. 43-45, Macmillan, London
2. Holum, J. Elements of General and Biological Chemistry, 2nd ed., 377, Wiley, NY (1968).
3. Martinek, R. Practical Clinical Enzymology: J. Am. Med. Tech., 31, 162 (1969).
4. Harrow, B., and Mazur, A.: Textbook of Biochemistry, 109, Saunders, Philadelphia (1958).
5. Pfeiffer, J. (1954) Enzymes, the Physics and Chemistry of Life, PG 171-173, Simon and Schuster, NY
6. Key stage 3/4 science: www.bbc.co.uk/education,
7. Internet source for pictures: www.google.co.uk,
8. Saronji T.Ramalingham B.Sc. (Hons), PhD: Modern Biology Textbook, PGs 229-230 & 484
9. P.W Freeland (1985): Problems in advanced level practical biology, Pg 32, Hooder and Stoughton, London, Sydney, Auckland and Toronto.
0. Cambridge Advanced Series: Biology 1, Mary Jones, Richard Fosbery, and Dennis Taylor.
Tolu Opawoye
