❌ 4. Required Practical: The Effect Of pH on The Rate Of Reaction Of Amylase (B3) Flashcards
What is an Independant Variable
An Independant Variable is the variable that is altered during a scientific experiment.
What is a Dependant Variable
A dependant Variable is the variable being tested or measured during a scientific experiment.
What is a Control Variable
A Control Variable is the variable that is kept the same during a scientific experiement.
Method for The Effect Of pH On An Amylase Reaction
Method:
- Use the syringe to place 2cm^3 of amylase solution into a test tube
- Use another syringe to add 1cm^3 of pH buffer solution to the test tube
- Place the test tube into a water bath set a 30°C and leave for 5 minutes
- Whilst waiting, add a drop of iodine solution into each dimple of a spotting tile.
- After 5 minutes, use another syringe to add 2cm^3 of starch to the amylase/buffer solution, start the stop clock and leave it on throughout the test. Mix using a plastic pipette.
- Remove a drop of amylase/starch/buffer mixture after 30 seconds (could make it 10 or 20) and add to the first drop of iodine on your spotting tile.
- Wait another30 seconds (could make it 10 or 20). Then remove a second drop of the mixture to add to the next drop of iodine.
- Repeat step 6 until the iodine solution and the amylase/buffer/starch mixture remains an orange colour.
- Record the time taken for the amylase to fully digest the starch.
- Repeat the whole process with a different pH buffer.
Equipment for The Effect Of pH On An Amylase Reaction
Equipment:
- Amylase Solution (0.5%)
- Starch Solution (0.5%)
- Iodine Solution in a dropper bottle
- Buffer solutions covering a range of pH values
- 5cm^3 syringes
- Pipette
- Test-tube rack
- Test tubes (one for each pH to be tested)
- Spotting tile
- Stop clock
- Marker pen or chinagraph pencil
- Water baths at 30°C
Heath and Safety for The Effect Of pH On An Amylase Reaction
Health and Safety:
- Use eye protection (goggles)
- Iodine is harmful - avoid contact with skin
- Make sure any pieces of glass don’t smash, meaning you could cut yourself
- Do not spill any any of the liquid solutions, if you do, then mop it up
What Should be done for Results?
You will need to calculate the rate of reaction at each pH level you have studied. To calculate the rate, use the formula:
Rate of Reaction = 1/Time
(Then design a suitable results table.)
My results table had three columns with ‘pH’ on the left, ‘Time(s)’ down the middle, and ‘Rate - 1000/Time (S^-1).
Under pH, I put each pH I tested (this when from 5.0 and went up 0.5 each time until it reached 8.0).
Under time, I put how long each pH took to break down all of the Starch.
Under rate, I did 1000 divided by the Time (s), to get my results.
What do you do with the Results and Results Table
From the Results and Results Table, I made 2 different graphs.
The first one was “How the pH level affects the time it takes for Amylase to break down Starch.” This had the pH level on the X-Axis and the Time (s) on the Y-Axis. This graph gave a down and the curved up line, with the optimum pH level sitting at around 7.
The second ome was “How the pH affects the rate of reaction for Amylse to break down Starch.” This had the pH level on the X-Axis and the Rate of Reaction (s^-1) on the Y-Axis. This graph gave a curving up to a point and then falling back down looking line, with the optimum pH level sitting at around 7.
What do you do with the Graph Result you have?
With a Graph, you can evaulate it if you are given a ‘Describe and Explain’ Question.
With a Describe and Explain question, you can separate it up into 2 different part. The describe part, and the explain part. The describing part is simply where you describe the line of the graph, as well as highlighting specific points and trends to do with the line itself, or the X and Y axes as well. In the explain, however, you have to go into a lot more scientific detail, and give reasons and explaination for and my ceratin this are happening and occuring in the graph. It is doing this explain well that will get the marks.
Describe and Explain for: How the pH level affects the time it takes for Amylase to break down starch
The graph that shows the how the pH affects the time (s) in which the enzyme amylase breaks down the molecule of starch follows a general trend of the time slowly declining, and then the time starts to increases again to create a sharp upwards turn. To look at this graph in more detail, we can see that at as the pH increases, the amount of time it takes for the starch to get fully broken down is shorter, which is shown by the fact that it takes 270 seconds for it to break down on pH5 and then on 150 seconds to do the same thing on pH6 (all at 30°C). The trend is followed until is stays around the same at pH6 and pH6.5 at a time of 150 seconds each. After this, there is a sharp decrease down, taking a short as 60 seconds (1 minute) on pH7, making it the sweet spot in this case, before it rises up to 300 seconds on pH8.
This will be because the level of pH will change or alter the active site of the amylase enzymes’s active site, meaning when an optimal pH is found, the active site will be in it’s most effective and efficient form/state. This will be why pH5 doesn’t get you the quickest possible time (270 seconds), because it is too acidic, as pH levels that are less than seven are acidic, while pH levels more than seven are basic, so each enzyme will react differently to different levels of pH. This will explain the gradual decrease in time on the, until it hits it sweet-spot. This sweet spot/optimum pH in particular is pH7 which finds itself in between the acidic and basic thresholds, taking the amylase enzyme only 60 seconds to completely break down the starch molecules. This means that the active site is in it’s best condition, therefore allowing it to be as successful and as effective as possible physically breaking down the molecules (meaning it has a higher success rate for each collision). However, as the pH increases, the environment it creates starts to become less acidic and more basic, which isn’t good for the enzyme, resulting in the fact that the active site begins to change it’s specific shape, resulting in it being less effective and efficient in collisions, which is why there is such a sharp increase in time taken on the graph, leaving pH8 on 300 seconds. This shows why the graph curves and travels in the way and direction that it does, and how the pH can affect the time of the reaction.
Describe and Explain for: How the pH affects the rate of reaction for Amylase to break down starch
The graph that shows how the pH affects the rate of reaction (s^-1 ) for the amylase breaking down the starch follows the inverse pattern of the time graph, with the rate of reaction increasing from pH5 (3.7 s^-1) to pH6 (6.7 s^-1), whilst pH6 is kept the same for pH6.5. Then there is a steep increase to the optimum pH of pH7 where the rate of reaction is 16.7 s^-1, and after this it then it quickly decreases to a rate of reaction being 0.3 at pH8.
This will be because the level of pH will change or alter the active site of the amylase enzymes’s active site, meaning when an optimal pH is found, the active site will be in it’s most effective and efficient form/state. This will be why pH5 doesn’t get you the best possible rate of reaction (3.7s^-1), because it is too acidic, as pH levels that are less than seven are acidic, while pH levels more than seven are basic, so each enzyme will react differently to different levels of pH. This will explain the gradual increase of the rate of reaction on the right, until it hits it sweet-spot. This sweet spot/optimum pH in particular is pH7 which finds itself in between the acidic and basic thresholds, with the rate of reaction at 16.7 s^-1. This means that the active site is in it’s best condition, therefore allowing it to be as successful and as effective as possible in physically breaking down the molecules (meaning it has a higher success rate each collision). However, as the pH increases, the environment it creates starts to become less acidic and more basic, which isn’t good for the enzyme, resulting in the fact that the active site begins to change it’s shape, resulting in it being less effective and efficient in collisions, which is why there is such a sharp decrease in the rate of the reaction on the graph to the right, leaving pH8 on 0.3s^-1. This shows why the two graphs curve and travel in the way and direction that they do, and how the pH can affect the rate of reaction.
How to get Gradients and Rates from Graphs
You can calculate the rate of reaction at each section of the graph by calculating the gradient.
This is by basically doing:
Rise/Run
This works on straight gradients, but if the gradient is curved, then you will need to get the tangent of the line, and essentially make it into a triangle before doing the Rise/Run
(Check Goonotes Workbook: “RP - The Effect Of pH on The Rate Of Reaction Of Amylase” for more detail and pictures).
What is the Independant, Dependant and Control Variable in this Experiement
The Independant Variable is the pH
The Dependant Variable is the Rate of Reaction
Some of the Control Variables are the Temperature, the volume of amylase, the starch solution and the buffer.
How does Temperature affect an Enzyme Reaction
The reactions that take place in cells happen at relatively low temperatures, as with other reactions, the rate of enzyme-controlled reactions increases as the temperature increases.
For most organisms this is only true up to temperatures of about 40°C. After this the protein structure of the enzyme is affected by the high temperature, as the long amino acid chains begin to unravel, and as a result, the shape of the active site changes. This means the substrate will no longer fit in the active site, as the enzyme is said to have been denatured and can no longer act as a catalyst, so the rate of reaction drops dramatically. Most human enzymes have an optimum temperature of 37°C (the human body temperature).
Without enzymes, none of the reactions in your body would happen fast enough to keep you alive. This is why it is dangerous if your temperature goes too high when you are ill. Once your body temperature reaches about 41°C, your enzymes will start to get denatured, resulting in death.
How does the pH affect an Enzyme Reaction
The shape of the active site of an enzyme comes from forces between the different parts of the protein molecule. These forces hold the folded chains in place. A change in pH affects these forces, which is why it changes the shape of the molecule/enzyme. As a result, the specific shape of the active site is lost, so the enzyme is no longer acting as a catalyst. Different enzymes work best at different pH levels. A change in pH can stop them working completely.
Different enzymes will be suited to different pH levels. For example, the optimum pH for Pepsin is 2, while the optimum pH for pancreatic amylase is 8.
