Full version Effect Of Temperature On An Enzyme Controlled Reaction

Effect Of Temperature On An Enzyme Controlled Reaction

This print version free essay Effect Of Temperature On An Enzyme Controlled Reaction.

Category: Science

Autor: reviewessays 01 January 2011

Words: 3517 | Pages: 15

To investigate the effect of temperature on an enzyme controlled reaction

Introduction and planning

For the investigation of enzymes, I am going to conduct an experiment to see how temperature can affect the rate of reaction of enzymes by testing it with starch.

The enzyme that we are going to use is called amylase. We are going to test this enzyme with starch. By mixing amylase and starch solutions together under different temperature conditions, we can record the rate of reaction by taking a sample out and test it with iodine solution to see if there is any remaining starch present.

We have to use the enzyme amylase because enzymes will only work on a specific substrate i.e. amylase will work on starch because of its special shape of active site. This is known as the ‘enzymes specificity’.

The enzyme amylase is used for hydrolysing starch and glycogen to form glucose and maltose. This enzyme is found in human saliva for the use of breaking down starch in food, enabling the body to absorb and produce ATP energy.

Enzymes are in all living organisms, they are proteins made up of polypeptide chains (which are made up of many amino acids) with irregular components which give it a globular shape. This makes it a dense, small, compacted molecule that can move around very easily.

For the enzyme to work, it must collide with a substrate. If they collide at the right place, the substrate will then ‘fit’ into the active site of the enzyme forming enzyme-substrate complex; fitting into the active site puts a strain on bonds in the substrate, so the substrate molecule breaks up more easily. In order for any reaction to occur, there must be energy to allow it to happen; this is called the activation energy. As shown below.

http://en.wikipedia.org/wiki/Enzyme_reaction_rate

This is a general graph that shows how activation energy works. The top dotted line to the time line is the activation energy needed for the reaction without enzymes; and the second dotted line to the time line is the energy needed to start a reaction with enzymes. This clearly shows that the differences between using an enzyme to start the reaction and not using it. The y axis represents two different things on this graph; the positive represents the used energy to start the reaction, and the negative half is showing the energy released for the reaction. Overall, the reaction with the enzymes can work faster then the other one without, but they still reach the same energy out-put at the same time forming same the products, also with the help of enzymes, the energy needed to start the reaction reduces compared with the energy needed without the enzyme.

There are many factors can affect the rate of reaction in enzymes:

• Temperature

• pH

• Concentration of enzymes

• Concentration of substrate

Temperature

This factor has a big influence on enzymes’ activity, because like any other chemical reaction, heat is needed to give the molecules more energy to move around – kinetic energy, so the molecules will move faster and therefore increase the chance of collision.

The following graph shows the general reaction rate of the enzymes against the temperature.

As the temperature increases, the reaction of enzymes also increases due to more kinetic energy; more vibrations, resulting an increased rate of collision with the substrate. But as it goes above the maximum temperature (optimum temperature), it starts to decrease rapidly. This is because the high temperature/ the high kinetic

energy breaks the bonds that hold the enzyme’s tertiary structure together which change the shape of the active site and will not react with the substrate anymore – denatured.

Denaturation is often irreversible.

pH

There is also an optimum pH value where the enzymes work most efficiently. Most enzymes work best around pH 7, which is neutral.

pH is a measure of hydrogen ions in a solution, if it is too acidic, the enzymes will become denatured, if the enzymes are too alkaline, the enzymes will too be denatured. The H+ and the OH- ions can cause chaos to the ionic bonds between each polypeptide chain in the enzymes (protein), which then break up and make a permanent change to its tertiary structure, and therefore, enzyme denatures.

The rate of reaction did not start at the point zero, it started around 3 and reached its optimum at around pH 7 to 8. It started to decrease and finally there were no reactions at around pH 11 to 12.

Concentration of enzymes

If the concentration of enzymes increases, there is an increase of enzyme molecules and active sites available. This gives a higher chance of collision with a substrate and forms a product.

The graph should look like this:

The rate of reaction is directly proportional to the enzyme concentration.

As long as there is enough substrate available to bind with an active site, the rate of reaction will increase linearly with enzyme concentration.

Concentration of substrate

As the concentration of substrate increases, the rate of reaction will also increase because the substrates are binding with enzymes. At the beginning of the reaction, the line increases steeply because there is less substrate compared to the concentration of enzymes. They will bind with an enzyme very quickly, resulting in a steep linear at the beginning. But as soon as the amount of substrate increases to a certain point (saturation point), the rate of reaction starts to flatten out. This is because there aren’t enough enzymes free for binding.

It will always get to a point where all the enzymes are being used and adding more substrate will have no effects on the rate of reaction.

By looking at the picture below, which I took from the Internet, the Lock and Key theory is about an area on the enzyme called the active site; this is an area which the substrate molecule fits into. The size and shape of the substrate matches exactly to a specific enzyme’s active site, so they fit together like a key fitting into a lock. This shows how an enzyme-substrate complex is formed.

In the picture, (a) is the substrate fitting into the active site

(b) is the enzyme-substrate complex.

(c) is the products formed by breaking the substrate and the enzyme is able to re-use again now.

This picture was taken from the website www.google.com by searching lock and key mechanism

Other than breaking the enzymes into two products, it can also put them back together.

This picture I took from the website www.tarleton.edu/ ~anatomy/enzyme.html is showing how two substrates get into the active site of the enzyme and form a peptide bond becoming a single dipeptide product.

During stage two to stage three, when they both meet, they create a peptide bond and give out a water molecule, and then the substrate breaks off and leaves the enzyme’s active site allowing the cycle to begin again.

Hypothesis

From studying the graphs on pages 1 and 2, I predict that as the temperature increases, the speed of reaction will also increase. After it has reached its optimum temperature, it will start to decrease dramatically and eventually stops reacting. I believe that the optimum temperature will be around 37°C, this is because the enzyme we are using are amylase, which is found in human saliva and the human body temperature is 37°C.

Enzymes can work faster when the temperature is higher; this is because the enzyme molecules are vibrating rapidly under high temperature, having a high kinetic energy, so it can collide with a substrate (starch) more often.

I believe that when the temperature reaches around 60°C, the enzymes activities will stop.

I can imagine at the end of the experiments, the results for 0°C will need a very long time for the amylase to break down starch. It may not even change colour in the spotting tile – staying blue-black. This is because under very cold conditions, the enzyme amylase works very slowly as there is very low kinetic energy, therefore slow movements, less successful collision of the active site and the substrate starch.

I believe the experiment for 20°C will not take as long as 0°C to react to break down starch. This is because it has a higher temperature.

I believe the experiment on 40°C will show the highest of all reaction rates, as it is close to the human body temperature and it should function normally, like in the body. The enzymes molecules and substrate molecules will be moving at a high kinetic energy level, the bonds between the enzyme molecules are just holding on.

And also for the last temperature 50°C, I think the rate of reaction will be very low, the spotting tile will mostly remain blue-black as nearly all the enzymes are now denatured; the high heat/ a very high kinetic energy breaks the hydrogen bonds betweens the enzyme, therefore enzyme molecules will loose their original shape, this means the enzymes are now denatured and will not binds with a substrate anymore to form a product.

Hydrogen bond is only a very weak attraction between two high electro-negatives, e.g. OH- and H+. But many of these attractions make the bond become very strong.

As long as I use the same concentration of substrate and enzymes, I will not have any problems on the effects of the concentration of substrates or enzymes – a control.

Variables

In this investigation, the variables that affect the activity of the enzyme were controlled so that they would not disrupt the success of the experiment:

Using same concentration of enzymes and substrate.

Using same volume of enzymes and substrate.

Using the same temperature for each experiment; 0°C, 25°C, 35°C, 45°C, 55°C.

Pressure is also a variable as it pushes the molecules closer together under a high pressure allowing the enzyme molecules and the substrates to collide more often. To avoid this, we have to try and stay at the same pressure at the time as we are doing the experiment, conduct the experiment in the same room at all times and try to do it at the same time as well

pH can cause a change in the reaction too; we can keep the pH at the same level if we add a buffer solution into the enzymes when mixing the substrate together.

Inhibitors – these are some of the major variables that can completely changes the rate of reaction, thus making the investigation unreliable. To control it, we must not add any inhibitors.

Safety

Clear the hallway; make sure there are no bags lying around on the floor.

Do not run around in the laboratory.

Clear the desks and put the chairs under the table.

Long hair should be tied back.

Iodine is toxic and an irritant and will stain clothing. Wear goggle to protect your eyes.

Be careful when using enzymes, as they are sometimes destructive.

Wear protective clothing.

If glass is accidentally broken, do not pick them up with bare hands.

When using a water bath, make sure you know the difference between hot and cold before you make contact with the water inside.

Steam can cause burns because it is extremely hot!

Control

A ‘control’ is needed so that we can study the detail by comparing results.

With a pipette, lift a drop of the starch solution from the test tube and mix it with the first drop of iodine in the first dimple in the tray. A blue/black colour should develop; this will be used as the control.

Only use a pipette for one solution, otherwise it will contaminate the sample you are using.

Apparatus:

Equipments Explanation

Substrate solution – Starch 1.0% Testing the experiment

Enzymes solution – Amylase 0.2% Testing the experiment

Iodine solution To test if there is starch present

Buffer solution Controlling the pH level

Spirit marker To label the test tubes, knowing which is which; in case of mixing up with other students.

Test tubes x15 Testing the experiment. Max to hold about 10cmÑ–

Boiling tubes x5 Testing the experiment. Max to hold about 30-40cmÑ–

Test tube rack Holding the test tubes

Measuring cylinder Measuring the exact amount of solution

Beaker x2 To create a stock solution

Goggle Safety reasons – Protecting eyes

Electrical water bath Creating a water bath that has a specific temperature – better accuracy then using a beaker.

Ice Creating a ice bath at 0-3°C

Stop watch Timing the reaction

Thermometer Measuring the temperature for the water bath and the solutions

Spotting tile Testing for result of starch’s present

Pipette x3 Pipette the solutions onto the spotting plate

I am using a different concentration of substrates and enzymes; this is because the school is providing this concentration.

Method

Please read before you start the experiment.

1. Always label the test tubes with your name and the contents, so that they don’t get mixed up with other students’ experiments.

2. Wear goggles throughout the experiment, clear hall ways for safety reasons.

3. Prepare the 1.0% of starch solution that we will be using.

4. Prepare the 0.2% of the enzymes solution that we will be using, in this case, we will be using the enzyme amylase.

5. Get out the water bath and set it to the desired temperature;

0°C, 20°C , 30°C, 40°C, 50°C

If we are starting from 0°C, this will have to be an ice bath, so ice cubes will be needed to keep the ice bath cool.

6. Measure 3cmі accurately with a measuring cylinder of 1.0% starch solution and put it in a test tube labeled “1”.

7. Measure 3cmі accurately with a measuring cylinder of 0.2% of amylase solution and put it in another test tube labeled “2”.

8. Place a thermometer into both solutions “1” and “2” and place the test tubes into the water bath setting the temperature that you will be experimenting on for. Eg. 0°C. (This will than allow the stock solution to acclimatise to the temperature wanted – more accurate.)

9. Wait until the temperature of the solutions are the same as the water bath.

10. While waiting, prepare the spotting tiles and iodine solution. On each dimple of the spotting tile, drop one drop of iodine solution ready for the test for the present of starch. Also get a few pipettes ready.

11. When the temperature of the solutions reaches the temperature of the water bath, mix the two solutions together and add buffer solution at the same time. Start the stop watch as soon as the two solutions are mixed. This will then be our stock solution.

By adding buffer solution, the pH of the mixed solution will be kept at the same value; therefore, one less variable and an easier control.

12. Take a drop of sample from the stock solution onto the spotting tile every 60 seconds with a pipette until the experiment has finished.

If the pipette comes into contact with the iodine solution when dropping, the pipette is now contaminated and needs to be changed.

13. Observe the colour. No starch breaks down as blue-black. Yellow-brown means starch is not present; therefore the breakdown of starch occurred.

14. Now repeat the experiment twice more to verify the results making a total of 3 results of the same temperature experiment. Then calculate the mean average.

15. Now repeat steps 5 to 15 with a different temperature.

16. End of experiment.

17. Rinse spotting tile with water and put it in the bowl for washing.

18. Pack everything up and place the used pipettes, test tubes and boiling tubes etc. in the bowl for washing.

I have chosen to repeat the experiment 3 times because this allows me to calculate an average time. This will ensure that there are no abnormal results and accuracy will be increased.

Pilot study

In order to make sure my experiment on “enzyme’s activity under different temperatures” works, I have conducted a pilot experiment.

This pilot experiment will enable me to improve the accuracy and the quality of the real experiment that I will be doing again in the future.

The pilot experiment was done as the method I have put down on the page before. But because of the lesson, I only had enough time for doing an experiment on one temperature, so I have chosen to do my trial on 40°C; this is because I predict that is the optimum temperature for the enzyme amylase to work the fastest.

I began my pilot experiment by preparing all the equipments I needed including putting the goggles on, and then I set the water bath to 40°C, carefully measured 3cmі of each enzyme and starch into a different test tubes by using a measuring cylinder and labelled them “1” and “2”.

Then I put them into the water bath allowing it to acclimatise to 40°C. Then I mixed them up and started the stop watch.

For every 60 seconds, I took a sample out and put it on the spotting tile which I prepared before with iodine solution on it.

After this experiment, I encountered many resulting in me making changes in order to improve the accuracy of my results.

In my pilot experiment, I have ended up with 16 same colour/ same looking colour – blue black as my result, which means the starch is present. I think the reaction has started but at a very slow rate. This may be caused by a small volume of solution, or the enzyme concentrations are just too little. I can improve this by increasing the volume of both enzyme solution and diluted starch solution to 10cmі and by diluting the starch solution with water. A test tube won’t be able to fit that amount of the solution, so I am going to use a boiling tube instead, which can take about 30 to 40 cmі maximum of solutions.

This will then result in a faster reaction, so therefore I also have to lower the time to collect the sample. I am going to change this time limit to every 30 seconds, so I can clearly see the changes to the colour on the spotting tile.

Apart from this problem, I also find difficulty in observing the colour of the mixed iodine solution by eye, as they all look the same. I can solve this problem by using a colorimeter, giving me a more accurate result.

A colorimeter measures the absorbance of radiation over a selected narrow range if wavelengths. The wavelength range is selected by choosing a filter which transmits light over the range absorbed by the compound under study. The wavelength used in this experiment is 390nm, which is the yellow colour’ wavelength.

Another problem will be the buffer solution. The school can not provide us with buffer solution and therefore the pH of the solution is not steady as we planned to be, and we can not do many things about this, so this becomes one variable that we need to beware of.

By doing the actual experiment instead of just think through it, I found out that there is some equipment I will need to add, remove or make changes to.

 When placing the test tubes in the water bath, there is nothing to hold the test tubes, so we need a rack to hold it up.

 For safety reasons, we should use a test tube holder to remove the test tubes out of the water bath as the steam can be extremely hot when operating with a higher temperature.

 A glass rode can be added to the apparatus for the use of taking a sample of the mixed solution. This will become more accurate because each drop will be about the same volume and it will not create bubbles as a pipette will.

 A beaker is not used, so we can remove it from the list.

 We need to label the test tubes more specifically, so not just “1” and “2”, but “1.0% starch”, “0.2% amylase” and “mixed solution”. We should have these ready before the experiment to increase the speed of doing the experiment.

 Control; we should add a drop of starch solution onto the spotting tile for comparison, and also an end point which is just iodine, so we know when the experiment is finished and when we can stop.

 Labelling the spotting tile is also useful. But start with zero by the dimple which just contains starch and iodine.

Variables noticed…

I have tried my best to avoid as many variables as possible. However, there are still same variables as there are things that we have no other way to control.

For example:

The water bath’s temperature; temperature can not always stay the same as it is operated by a build-in thermostatic control, it will keep heating the water until it can detect a raise in temperature and it will automatically switch itself off and allow the temperature to drop back down to the set temperature. This means there will be a constant change in temperature.

Also, the concentration of the enzymes and starch are not the same because the school can only provide us with that concentration. But as long as the percentages are in the same proportion, I think it will still be alright.

pH is also a variable that we need to take into account of, because we can not use the buffer solution as planned, therefore the pH might change during the reaction.

The timing doesn’t start exactly when the solutions are mixed and the action of taking sample from the stock solution to the spotting tile are not exactly spot on, this is because of the procedure of taking a sample by using a glass rod can not be 0 seconds.

Pressure is also a important factor that we can not control in school, so we will have to do all the experiment in the same room, same place and even need to be doing it in the same time!

Inhibitors are also a factor that will have an effect on the rate of reaction, so we will not add any inhibitor into the solution just to complicate things.