Effect+of+Temperature+and+pH+on+Enzyme+Action+Experiment


 * Partner: Jiale

Apparatus & Materials:** 1. Bunsen burner, tripod stand and gauze 2. 3 water baths 3. Thermometer 4.12 test tubes and rack 5. Measuring cylinder 6. 4 250 ml beakers 7. 2 white tiles 8. 2 droppers 9. Ice cubes 10. Distilled water 11. Test tube holder 12. Stop watch 13. Goggles 14. 2% starch solution 15. 1% amylase solution 16. 1 M Hydrochloric acid solution 17. 1 M Ammonium Hydroxide solution 18. iodine solution

1. Label 8 test tubes A1, A2, B1, B2, C1, C2, D1 and D2. 2. Add 5cm3 of starch solution to tubes A1, B1, C1 and D1. Add 3 cm3 distilled water to D2, and then 3 cm3 of amylase solution into tubes A2, B2 and C2. 3. Place A1 and A2 into a beaker of ice water, B1 and B2 into a water bath at 370C, and D1 and D2 into another water bath at 370C. 4. Place C1 and C2 in boiling water. 5. Leave the tubes for 10 minutes to allow the solutions in the tubes to reach the temperature of the water bath. 6. Pour the contents of A2, B2, C2 and D2 into the starch tube, A1, B1, C1 and D1, next to it. Record the time immediately. Keep the tubes A1, B1, C1 and D1, containing the mixture in the respective water bath throughout the experiment and ensure that the temperatures are maintained. 7. After 10 minutes, test the mixture in each tube with iodine solution, on a white tile. Draw table 1 with appropriate headings and record the results and conclusions.
 * Procedure:**
 * PART A**

1. To each of the 4 test tubes, W, X, Y, Z, add 2 cm3 of starch solution. Place the tubes in a water bath at 370C. 2. Make up the contents of the 4 test tubes as shown in the table below. 3. Test the solution with iodine solution at the beginning of the experiment and after 10 minutes.
 * PART B**

__**Part A: Investigating the Effect of Temperature on Enzyme Action**__

To find out the effect of temperature on enzyme action.
 * Aim**


 * **Test tube** || **Contents** || **Surrounding Conditions** || **Iodine test** || **Conclusion** ||
 * A1 || 5 cm3 of starch solution + 3 cm3 amylase solution || Placed in a beaker of iced water || Blue-black || Starch is present in the solution ||
 * B1 || 5 cm3 of starch solution + 3 cm3 amylase solution || Placed in a water bath at 37 degrees Celsius || Yellow-brown || Starch is not present in the solution ||
 * C1 || 5 cm3 of starch solution + 3 cm3 amylase solution || Placed in boiling water || Blue-black || Starch is present in the solution ||
 * D1 || 5 cm3 of starch solution + 3 cm3 distilled water || Placed in a water bath at 37 degrees Celsius || Blue-black || Starch is present in the solution ||

Tube B1 showed that starch digestion has occurred, whereas tubes A1, C1 and D1 contained solutions that gave a blue-black colour. The solution in tube B1 showed that starch digestion has occurred, as when the iodine test was carried out on it, it remained brown in colour, with no visible colour changes. This shows that starch is not present in the solution, as compared to the three other test tubes with solutions that turned blue black as they contained starch. Also, the purpose of Tube D was to act as a control of the experiment.
 * Interpreting the Results**

The results of this experiment may be due to the fact that as the temperature rises, the reacting enzyme molecules move faster. Particles of matter are in constant random motion, so are the reacting molecules (enzyme and substrate molecules) which move and collide with one another. Temperature increase results in the increase in kinetic energy, which causes the reacting molecules to move around faster and thus the enzyme and substrate molecules collide with each other more often.Thus the rate of formation of the enzyme-substrate complex increases and the formation of the product also increases. Therefore, this explains why the solution in tube C remained brown as starch was digested very quickly as when placed in a temperature of 37 degrees Celsius, the enzymes gain kinetic energy and are more active. However, the solution in test tube C turned blue black as the starch was not digested yet. This is because enzymes are made of proteins, and during high temperatures, the chain of amino acids from the proteins denature. The coils are folded up and held in place by weak hydrogen bonds. An increase in temperature results in violent vibrations which causes it to lose its shape and active site, thus destroying the protein and the enzyme can no longer act as a catalyst. Whereas, the solution in tube A turned blue black as the starch was not fully digested due to the fact that the enzymes were not active, due to low temperatures and thus low kinetic energy for the enzymes.

The less time taken to digest starch, the more active the enzyme is. Thus, we can conclude that the higher the temperature, the more active the enzymes will be and thus the faster the rate of chemical reaction. However, high temperatures will cause enzymes to denature and thus not being able to act as catalysts as they are destroyed by the high temperatures.

__**Part B: Investigating the Effect of pH on Enzyme Action**__

To find out the effect of pH on enzyme action.
 * Aim**

After: Iodine turns blue black || Starch was present in the solution throughout the experiment || After: Iodine remains unchanged at brown || Starch was not present throughout the experiment || After: Iodine remains unchanged at brown || Starch was not present throughout the experiment || After: Iodine turns blue black || Starch was present in the solution throughout the experiment ||
 * **Test tube** || **Contents** || **Iodine test** || **Conclusion** ||
 * W || 2 cm3 of starch solution + 2 cm3 of HCL + 2 cm3 of amylase || Before: Iodine turns blue black
 * X || 2 cm3 of starch solution + 2 cm3 of distilled water + 2 cm3 of amylase || Before: Iodine remains unchanged at brown
 * Y || 2 cm3 of starch solution + 2 cm3 of NH4OH + 2 cm3 of amylase || Before: Iodine remains unchanged at brown
 * Z || 2 cm3 of starch solution + 4 cm3 of distilled water || Before: Iodine turns blue black

Tubes X and Y showed that starch digestion has occurred even before the 10 minutes was up, whereas tubes W and Z contained solutions which gave a blue-black colour. The solution in tube X and Y showed that starch digestion has occurred, as when the iodine test was carried out on it, it remained brown in colour, with no visible colour changes. This shows that starch was digested, as compared to the other two test tubes with solutions that turned blue-black as they contained starch.
 * Interpreting the Results**

The results may be due to the fact that enzymes are affected by the acidity of the alkalinity of the solution. Some enzymes work better in slightly acidic, or slightly alkaline solutions. The solution in tube Y remained brown in colour after the iodine test, as ammonium hydroxide, being a weak alkaline, causes the enzyme to be more active, and the starch an be digested quickly. As for test tube W, HCL is a strong acid, and thus denaturing the enzyme due to the fact that extreme changes in acidity or alkalinity of the solutions denature the enzymes. When a pH increases, the charges on the substrate neutralised, and an extreme increase in pH breaks the hydrogen bonds in the enzyme molecules, The enzyme changes it shape and loses its active site, therefore becomes denatured. Also, when a pH decreases, it changes the charges in the active site. The enzyme and the substrate repel each other as they have the same charges. Extreme decrease in pH would cause enzyme action to slow down or even stop. Therefore, enzyme action is slowed down/stopped in tube A as hydrochloric acid is a strong acid.

In conclusion, slightly acidic or alkaline solutions may help enzyme action, however, extreme changes in the acidity or alkalinity may cause the solutions to denature, and stop enzyme action.