Group 4 Chemistry - Amylase

Part 2: Temperature of Surroundings

Part 3: pH of Surroundings

Dependant

All Parts: Rate of Reaction

Materials

• Distilled Water                                                        
• 0.5% Starch Suspension (Made up in 0.25M NaCl)
• pH Buffer 3, 7, 5 and 9
• IKI (Iodine in Potassium Iodine)
• Saliva (5mL)

Apparatus

• Standard Glassware
• Safety Goggles/ Aprons
• Pipettes
• Hot Plate
• Thermometer
• Well Plates
• Stopwatch

Method

Appropriate safety precautions must be taken at each stage throughout the experiment. Eye Protection should be worn at all times as well as aprons. Special care should be taken when handling the pH buffers as well as the IKI.

First preparations for the 3 different phases of the experiment must be made. Saliva must be stimulated and collected in a beaker. Approximately 5mL should be collected. Dilute solutions of saliva should then be created in the ratios as follows. Place each solution in a small beaker.

 

Part 1. Reaction Rate against Enzyme Concentration.

1. 10 drops of each Saliva Solution should be transferred into 4 labelled test tubes. Be sure to use a clean pipette for each concentration.
2. 2mL of Starch Solution should then be transferred into each of another 4 test tubes. 2mL of pH buffer 7 should also be added to each of these test tubes.
3. Place both sets of test tubes in a water bath at around 37˚C. Let them sit in the water bath for 5 minutes.
4. Prepare a Well Plate with several wells filled with IKI solution.
5. Pour the contents of one of the starch tubes into the 10% saliva. Swirl to mix and immediately return the test tube to the water bath. Begin timing as soon as the test tubes have been mixed.
6. Test the solution for starch by dropping a small amount into one of the wells of IKI. Observe any change in colour, a change means starch is still present.
7. Repeat the test with a fresh drop of the solution every 5 seconds until the colour of the solution remains the same as the IKI. Record the time the colour of the solution changes. If after 10 minutes there has been no change, record the reaction time as no reaction.
8. Repeat steps 5, 6 and 7 using the other concentrations of saliva.

Part 2. Reaction Rate against Temperature.

1. 10 drops of the 2% saliva solution should be transferred into 5 test tubes. Each should be heated to different temperatures. Temperatures should be roughly as follows:

1. 0˚C -10˚C
2. 20˚C -30˚C
3. 37˚C
4. 45-55˚C
5. 70˚C-100˚C

2. 2mL of Starch Solution should then be transferred into each of another 5 test tubes. 2mL of pH buffer 7 should also be added to each of these test tubes. These test tubes should also each be subject to one of the 5 temperatures.
3. Prepare a Well Plate with several wells filled with IKI solution.
4. After approximately 5 minutes have passed at these temperatures, pour the contents of one of the starch solution test tubes into its temperature corresponding amylase test tube. Take the temperature of the solution immediately after mixing. Swirl to mix
5. Test the solution for starch by dropping a small amount into one of the wells of IKI. Observe any change in colour, a change means starch is still present.
6. Repeat the test with a fresh drop of the solution every 5 seconds until the colour of the solution remains the same as the IKI. Record the time the colour of the solution changes. If after 10 minutes there has been no change, record the reaction time as no reaction.
7. Repeat steps 4, 5 and 6 using the other temperatures

Part 3. Reaction Rate against pH

1. Use narrow pH paper to determine the pH of the Saliva collected.
2. 10 drops of the 2% saliva solution should be transferred into 4 test tubes. Place these test tubes in a water bath of approximately 37˚C.
3. 2mL of Starch Solution should then be transferred into each of another 4 test tubes. In each of the 4 test tubes 2mL of a different pH buffer should be added. pH buffers 3, 5, 7, 9 should be used. Label these test tubes appropriately. Place these test tubes in a water bath of approximately 37˚C.
4. Prepare a Well Plate with several wells filled with IKI solution.
5. Pour the contents of the pH 3 solution 2% saliva solution. Swirl to mix and immediately return the test tube to the water bath. Begin timing as soon as the test tubes have been mixed.
6. Test the solution for starch by dropping a small amount into one of the wells of IKI. Observe any change in colour, a change means starch is still present.
7. Repeat the test with a fresh drop of the solution every 5 seconds until the colour of the solution remains the same as the IKI. Record the time the colour of the solution changes. If after 10 minutes there has been no change, record the reaction time as no reaction.
8. Repeat steps 5, 6 and 7 with different the different pH solutions.

Results

Part 1. Reaction Rate against Enzyme Concentration.

Part 2. Reaction Rate against Temperature.

Part 3. Reaction Rate against pH.

Conclusion

The three parts of the experiment have led to good results that we can use to validate our hypothesis. The results clearly show that as enzyme concentration increases to rate of reaction also increases. This is because.

For Part 2 of the experiment we also gained excellent results. The results gained concur with the prediction made and further validate the hypothesis. The greatest rate of reaction was that of test tube placed under a temperature of 37˚C. This is due to the enzyme’s optimum operating conditions being that of the human body, the human body having a fairly consistent temperature of 37˚C. The other results show that as the temperature moves away from the optimum operating temperature the reaction rate decreases until the point where the enzyme becomes completely denatured and no longer will break down the starch solution. As the enzyme is heated or cooled the protein loses its shape and thus causes the loss in activity and the eventual denaturing when the protein has changed in shape to such a degree that it can no longer perform its original process. I can compare my graphed values to that of a graphed value found elsewhere, it is clear that there is a similar trend.

For Part 3 of the experiment the results gained were also good. These set of results also confirmed the predictions made at the beginning. The greatest rate of reaction was that of test tube with a pH buffer of 7. This is due to the enzyme’s optimum operating conditions being that of the mouth, the mouth has a fairly constant pH of 7. The other results show that as the pH moves away from neutral the rate of reaction decreases until the point where the enzyme becomes completely denatured and no longer will break down the starch solution. As the enzyme is exposed to high or low pH the protein gains either a large negative charge or large positive charge respectively. This in turn causes the unfolding of the protein as the like charges repel, this unfolding causing the protein to lose its function. I compared my results to that of literature values and also compared my graph to an accepted graph. Correlation can be seen in both.

Therefore we can answer the research question and say yes there that pH, temperature, and concentration do have an effect on the reaction rate of salivary amylase in the breakdown of starch.

Evaluation

The method and materials are proven to be good as the results garnered are good and validate earlier predictions. There are experimental uncertainties and these have been included within the results tables above. However these uncertainties have little effect upon the experiment as no calculations were made with the results. The way the timing was done meant that the uncertainties had little effect. However we must also take into account these uncertainties, they could be the cause of some errors that have led the results to be slightly off the accepted values.

The data gained does not present a smooth curved graph as the accepted values show. Doing more trials would have given us a more comprehensive set of data. This would have created a better graph and would have given us greater information to draw conclusions. These different trials could have been of different pH values, temperatures, and concentrations over a broader spectrum.

Another improvement to the experiment would be to repeat the same experiment with a more enzymes to compare the varying effect of pH, temperature and concentration on other enzymes. Examples of other possible enzymes can be found in the table of values found in the conclusion. It would also be a good idea to compare the different form of amylase, possibly using concentrated amylase rather than that found in saliva. We could then calculate the exact concentration of amylase.

A change in timing method could yield more accurate results, we are working with 5 second intervals, if we could reduce this we would have more accurate results.

However the experimental design, procedure and materials have given excellent results and have validated the prediction, nothing needs to be changed to answer the research question.

Sources

http://www.coolschool.ca/lor/BI12/unit7/U07L03/

http://www.worthington-biochem.com/introbiochem/effectspH.html