The Effect of the Concentration of an Inhibitor on The time taken for the Enzyme to Fully Breakdown the Substrate

Aim

For this investigation I am going to investigate the effect of the concentration of an inhibitor, on the time taken for the enzyme to fully breakdown the substrate.

Introduction

I am going to complete my aim by planning an experiment, carrying out the experiment, recording any relevant results and plotting graphs from which I will be able, hopefully to gain a strong conclusion. I will finally evaluate the whole investigation.

I will be using human amylase to breakdown the substrate starch. The reason this enzyme has to be used is because each enzyme is designed specifically to break down only one substrate as each enzyme is made of a protein that causes it to be a specific shape, in this case the enzyme Amylase can only break down starch. Also the inhibitor I will be using is copper sulphate, a heavy metal ion.

Background Reading

Amylase, like other enzymes, works as a catalyst, i.e. it is unchanged by the reaction, but makes the reaction easier by reducing the energy required for it to happen. Catalysts speed up the reaction. The theory behind the working is called the "lock and key" theory: the enzyme is shaped so that the products fit into them, react and are released. Amylase digests starch by catalysing hydrolysis, which is splitting by the addition of a water molecule. Therefore starch plus water becomes maltose (which is equivalent to two joined glucose molecules).

There are two kinds of amylase enzymes. Alpha-amylase is found in saliva and is called ptyalin. This can carry on working in the stomach for several hours (and can digest up to 40% of starch under correct conditions of stomach acidity and food solidity). The other kind is called pancreatic amylase and is secreted in pancreatic juice, into the small intestine or ileum.

Inhibitors are chemicals, which reduce the rate of an enzyme-catalysed reaction, and they do so by altering the shape of the active site. They can be divided into competitive (competes with the substrate for the active site) and non-competitive (alter the shape by binding to the enzyme away from the active site). They can also be defined by if there effects are permanent or non-permanent. Competitive enzymes are never permanent; however not all non-competitive inhibitors are permanent.

Prediction

My prediction is that as the concentration of the inhibitor present increases the length of time for the reaction to be complete would increase thus decreasing the rate of the reaction

Hypothesis

As I am using copper sulphate, the inhibitor alters the shape of active site non-competitively. This means that it does not attach to the active site but attaches to the enzyme elsewhere altering the shape of the active site this way. Copper sulphate effects are also permanent which means that the enzyme is unable to carry out further catalysis after coming in contact with the heavy metal ion (copper sulphate).

Therefore the presence of an inhibitor will lead to a reduced rate of reaction. The increase of concentration would mean that there would be a greater number of heavy metal ions present in the same volume, thus resulting in more encounters between the inhibitor and the enzyme. Which would render these enzymes useless, resulting in less enzymes being able to break down the substrate, which leads to a slower reaction rate.

Variables and Constants

In this investigation, the variables that affect the activity of the enzyme, Amylase, were considered and controlled so that they would not disrupt the success of the experiment. Because the factor I am trying to test is the effect of the concentration of the Inhibitor, I will be only changing the concentration of the inhibitor and nothing else in each test. This will make all of the tests identical (apart from the inhibitors concentration), which means the experiment should be accurate and fair. Therefore any trends in the results of my experiment I will be able to ...

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Variables and Constants

CONSTANTS

Temperature

As temperature increases, molecules move faster (kinetic theory). In an enzyme catalysed reaction, such as the decomposition of starch, this increases the rate at which the enzyme and substrate molecules meet and therefore the rate at which the products are formed. As the temperature continues to rise, however, the hydrogen and ionic bonds, which hold the enzyme molecules in shape, are broken. If the molecular structure is disrupted, the enzyme ceases to function as the active site no longer accommodates the substrate. The enzyme is denatured. To control this variable, the temperature was maintained at a fairly constant level that allowed the enzyme to work effectively (approximately 40ºC). This was achieved by a using a water bath to keep it at a constant temperature. It was also allowed a time of acclimatisation

Any change in pH affects the ionic and hydrogen bonding in an enzyme and so alters it shape. Each enzyme has an optimum pH at which its active site best fits the substrate. Variation either side of the optimum pH results in denaturation of the enzyme, and a slower rate of reaction. In this experiment, the pH was kept constant using a pH 7 buffer, selected to maintain a pH level suited to the enzyme by being equal to the natural environment of the enzyme (human tissue)

Substrate Concentration

When there is an excess of enzyme molecules, an increase in the substrate concentration, produces a corresponding increase in the rate of reaction. If there are sufficient substrate molecules to occupy all of the enzymes´ active sites, the rate of reaction is unaffected by further increases in substrate concentration as the enzymes are unable to break down the greater quantity of substrate. To control the substrate concentration, identical quantities of the substrate were used for each reading from 1% solution of starch. To ensure that this was measured precisely, 5ml syringes were used to accurately gauge to exact quantities

Enzyme Concentration

This was kept at a constant so that it would not affect the results, as an increase in the enzyme concentration would lead to a corresponding rise in the rate of reaction. To control the enzyme concentration identical quantities were used for each reading from 1% solution amylase using a 5ml syringe.

CONTROLLED VARIABLE

Inhibitor Concentration

An increase in Inhibitor concentration will lead to a corresponding decrease in rate of reaction. Where the inhibitor is limiting the rate of reaction. I varied the inhibitor concentration by altering the mass of copper sulphate in 100ml. I decided to use a permanent inhibitor (heavy metal ion) so that the temporary effect of reversible inhibitor does not affect my results.

Apparatus Needed

Test Tube Rack
4 x 5ml syringes
1 x10ml syringe
12 x 10 mm diameter Test Tubes
A stop clock
A 250ml Beaker
Thermometer
Ph 7 Buffer
Spotting Tile
Bunsen burner (with Tripod and gauze)
Iodine (with pipette)
Stirring Rod
Kitchen towel
A scale
8 x 15 ml beakers
Tongs

Reason for Equipment

15 x 10mm diameter test tubes will be used for mixing the amylase with the starch also copper sulphate and pH buffer in the water bath.

The test tubes have to be clean to prevent any unwanted contaminants getting into the experiment. 4x 5ml and 1 x 10 ml (for water) syringe used for very accurate measuring of the amylase, starch solution, water, and copper sulphate. This will be vital for getting the correct volume of the 3 substances into the test tubes and the correct amount of water for dilution.

Thermometer, the most accurate way of measuring the temperature of the water bath. Therefore the temperature of the enzyme and the substrate as well.

Stopwatch to measure accurately the time it takes for amylase to break down the starch.

250ml beaker Used for the water bath because it is large enough to hold the test tubes and a large amount of water

A scale to accurately measure the mass of substances to 1 millionth of a gram

Tongs to prevent my own body heat affecting the results

Procedure

First you must prepare your solutions of Differing concentrations for copper sulphate

Ranging from 0.01 through to 0.06 moles. You do this by placing a fraction 249g (relative atomic mass of copper sulphate plus five waters) in 10ml to achieve the required concentration.

Then you must prepare the 1% starch solution and the 1% amylase solutions that are simply 0.1g in 10ml of water.

Then syringe 2ml of each solution into test tubes including 2 ml of the pH buffer, to prevent contamination remember to use different syringes for different substances and to wash out the syringe when using a different concentration of copper sulphate from the previous experiment. Label and place in the test tube rack.

Prepare the apparatus as shown below in the labelled diagram

Test tube

Rack

Stop clock

Tripod

With gauze

And Bunsen

Burner HEAT spotting tile

And beaker

above

Heat the water bath to 40ºC. When achieved place test tubes of amylase and starch and allow the solutions to acclimatise for 10 minutes then apply copper sulphate and begin spotting with stirring rod placing samples of solution into the iodine in the spotting tile. Do this every minute until the initial colour change ceases to reoccur.

Method

First I prepared the Copper Sulphate using the scale to measure the mass needed for the different concentrations of copper sulphate.

Mass concentration

0.0249g 0.01 moles in 10ml

0.0498g 0.02 moles in 10ml

0.0747g 0.03 moles in 10ml

0.0996g 0.04 moles in 10ml

0.1245g 0.05 moles in 10ml

0.1494g 0.06 moles in 10ml

I placed the measured masses in 10 ml of water and stirred them until all the blue sediment had dissolved into the water, leaving a transparent blue solution.

Then I prepared the 1% starch solution and the 1% amylase solution again using the scale to measure the required mass. For Starch I warmed up the 100ml of water that it was dissolved in to ensure that it would mix. However for Amylase I just used cold water.

After that I syringed 2 ml of starch and 2 ml of amylase into separate test tubes from the made solutions of each. I then proceeded to syringe 2 ml of the selected concentration of copper sulphate i.e. 0.01 moles in to a test tube. And syringed 2 ml of the pH buffer into a separate test tube. I used different syringes for each solution to prevent contamination. And I labelled them.

Next, I set up the apparatus, and heated a water a bath to approximately 40 degrees Celsius and placed a test tube of starch and one test tube of amylase I started the stop clock and left them to acclimatise for 10 mins

While the starch was acclimatising I placed droplets of iodine in the spotting using a pipette.

Once the starch and amylase was acclimatised I mixed them together and added the buffer and the copper sulphate

I then used the rods to test for starch by placing it in the solution and then removing it and then placing it on one of the spots on the spotting tile. I then wiped clean the rod with the kitchen towel to prevent contamination. I then repeated this process every minute using a different spot each time until the colour change did not take place anymore.

Observations and Measurements

In order to decide how varying the inhibitors concentration affected the decomposition of starch, the rate of reaction was measured. To do this accurately, the time taken for the starch not to be detectable present was determined. This was achieved by observing the time taken for the change in the colour of iodine to cease. This was an accurate measure of how the inhibitor concentration influenced the breakdown of starch, as the time taken for this take place is dependent on the rate of reaction. The marked points remained the same distance apart for each reading for different inhibitor concentrations so that they could be accurately compared and trend observed. All measurements were taken so that the stopwatch was started once the substances were mixed and the stopwatch stopped once the iodine remained orange. This was the same for every reading.

Data handling

The data obtained from this investigation has been recorded in a table showing the time, enzyme concentration and rate of reaction. This means that the results of the experiment are presented in a clear and orderly fashion that allows patterns in the results to become more obvious. The rate of reaction was calculated by dividing 1000 by the time taken for the colour change to cease. By calculating the rate of reaction instead of merely using the time readings, the quicker reactions will be represented as a greater value for the rate of reaction rather than a small time value. This makes the graphs more clear and easier to analyse. Patterns within the results collected from the experiment, are best shown on a graph. This is because overall trends between the inhibitor concentration and rate of reaction can be portrayed more effectively and become more obvious

Limitations and Precautions

I monitored the temperature using a thermometer to ensure that it remained constant and not disrupt the results of the experiment by affecting the activity of the amylase

A pH buffer was used to maintain a consistent pH level in the test tubes. This way there was no variation in pH that might have resulted in an increase or decrease in the rate of reaction.

A major limitation of this investigation was the time. It meant that only 6 different inhibition concentrations could be measured at intervals 0.01 moles. This means that only very general, overall trends can be identified across the results. Patterns between these values can only be approximated and are not necessarily accurate.

Safety

Laboratory coats were worn during the investigation to prevent chemicals from spoiling clothes. Safety aspects Goggles must be worn and especially while using the iodine as it can be irritable to the eyes. Stand up while using the Bunsen burner so you can move away quickly should the water bath fall. Any long hair tied back to keep out of way of flames.