Investigating the effect of pH on amylase activity

In a nutshell

There are several factors that affect the activity of enzymes. Amylase is an enzyme that breaks down starch into a smaller sugar called maltose. By using amylase to break down starch at different pH values, you will be able to investigate the effect of pH on enzyme activity. You will do this by measuring the pH of different solutions and using iodine solution as an indicator of the breakdown of starch.

Equations

After an experiment, it is useful to calculate the rate of reaction. For this practical, the following equation can be used to calculate the rate.

Word equation	symbol equation
$rate = {1000 \over time}$	$R = {1000 \over t}$

Variable definitions

quantity name	SYMBOL	unit	Unit symbol
$rate$	$R$	$per\space second$	$s^{-1}$
$time$	$t$	$second$	$s$

Equipment list

The following equipment list can be used to conduct an experiment to evaluate the effect of pH on amylase activity.

equipment	use
$10$ test tubes	To hold the starch, amylase and buffered solutions.
Test tube rack	To hold the test tubes.
Water bath	To keep reacting solutions at a constant temperature.
Spotting tile	To put the iodine and reacting solutions into.
$5\space cm^3$ measuring cylinder	To measure various solutions.
Pasteur pipettes	To add the amylase solution to the mixture solution.
Glass rod	To add drops of the mixture solution onto the spotting tile.
Stopwatch	To measure the time taken.
Starch solution $(1\%)$	The polysaccharide that is broken down by starch.
Amylase solution $(0.1\%)$	The enzyme that digests starch.
Labelled buffered solutions at a range of pH values	To investigate the activity of amylase at different pH values.
Labels	To label the relevant test tubes.
Iodine solution $(0.01\space mol/dm^3)$	The indicator of the breakdown of starch in this experiment.
Thermometer	To measure the temperature of the reacting solutions.

Experiment 1: Investigating the effect of pH on the rate of reaction of amylase enzyme

Experimental variables

All experiments have three different variables: the independent, dependent and control variables. The independent variable is the one you change. The dependent variable is the one that depends on what has been changed, therefore it is the one that you measure. The control variable is one you keep the same during the experiment.

Independent variable	$The\space pH\space of\space the\space buffered\space solutions$ .
Dependent variable	$The\space time\space taken\space for\space the\space reaction\space to\space be\space completed$ .
Control variable	$The\space concentration\space and\space volume\space of\space the\space amylase,\space iodine\space and \space starch\space solution\space used$ .

Safety precautions

When carrying out experiments, it is very important to consider safety precautions. This is so you and others do not get hurt.

hazard	risk	safety measure
Iodine solution	Iodine solution may cause eye irritation.	Wear safety goggles when handling the iodine solution.

Method

This is your instructions on how to complete the experiment.

1.	Heat the water bath to $35\degree C$ . Put $2\space cm^3$ of each buffered solution into separate test tubes, making sure to label each tube with the correct pH.
2.	To five test tubes, add $4\space cm^3$ of starch solution. Into another test tube, add $10\space cm^3$ of amylase solution. Make sure to label the relevant test tubes with 'starch' or 'amylase'.
3.	Put the test tube rack with the test tubes into the water bath. Place the thermometer in one of the starch test tubes to monitor the temperature. Allow the solutions to reach a temperature of $35\degree C$ .
4.	Put one drop of iodine solution into each depression on the spotting tile. To the first depression, also add one drop of starch solution. This will act as a comparison of colour the test buffers. Note: When mixed with iodine, starch gives a blue-black colour. When all the starch has broken down, it turns back to brown.
5.	Once the correct temperature has been reached, add $2\space cm^3$ of the first pH buffered solution to one of the test tubes of starch. Stir this mixture with a glass rod.
6.	Add $2\space cm^3$ of amylase solution to the mixture using a pipette. As soon as you add the amylase, start the stopwatch. Continue stirring the mixture.
7.	After $10\space seconds$ , add one drop of the mixture onto the second depression in the spotting tile using the glass rod. Then, rinse the glass rod with water.
8.	Every $10\space seconds$ , add a drop of the mixture to another depression in the spotting tile. Keep repeating this every $10\space seconds$ until the iodine does not change colour (i.e. remains brown). Note: Remember to rinse the glass rod with water before adding another drop of the mixture to the spotting tile.
9.	Record the time taken for the amylase to completely break down the starch.
10.	Repeat steps 5 - 9 with all of the other pH buffered solutions.

Analysis

This is how you can use your data to form conclusions on your experiment.

1.	Firstly, you will now have a table with a list of pH values and the time taken for amylase to completely break down the starch.
2.	For each pH value, calculate the rate of reaction. $rate = {1000\over time}$
3.	You should then plot the rate and the pH onto a graph. The rate of reaction should be on $y$ -axis as it is based on the dependent variable. The pH of the solution should be on the $x$ -axis as it is the independent variable. Draw a line through the points on the graph.

Conclusion

Your graph should show the optimum pH for the activity of the amylase enzyme. At this pH, the iodine solution will go from blue-black, back to its original brown colour in the shortest amount of time. This is because the enzyme will be the most efficient at this pH. There will be a gradual loss of enzyme activity away from the optimum pH, as the active site will become distorted. The graph will also show that, at extreme pH values furthest from the optimum pH, there will be no rate of reaction. This is because the amylase enzyme will be completely denatured at this pH and be inactive. The shape of the graph should be similar to the graph below.

Biology; Biology practicals; KS4 Year 10; Investigating the effect of pH on amylase activity — 1. At extreme pHs the rate of reaction is very low, 2. Optimum pH, 3. Enzyme denatures above the optimum pH.

Evaluation

Once you have completed your experiment, it will be important to consider the quality of your data and how accurate your results are. Identify potential sources of random or systematic error and suggest possible improvements and further investigations.