ENZYME

INVESTIGATION

AQA Biology Dual Award

Skills Tested:

Observation   /8

Analysis          /8

Evaluation      /6

The Skinners’ School

1.  Introduction

Enzymes were discovered by a German chemist called Eduard Buchner near the end of the nineteenth century. He discovered enzymes while trying to extract yeast fluid for medicinal use when he noticed that the sugar was continually being converted into alcohol. Even though Louis Pasteur had shown this about twenty years before Eduard Buchner, Louis Pasteur had said that it was the yeast itself that made the conversion.

Eduard Buchner said that it was not the yeast but the ‘juice’ inside them. The word enzyme was used for the active ingredient in the yeast juice. Enzyme literally means ‘in yeast’ ( ‘en’ = in, ‘zyme’ = yeast) and all enzymes are used as organic catalysts to speed up chemical reactions in organisms.

Without enzymes reactions in cells would be so slow that they would virtually not take place.

How enzymes work

Enzymes work very quickly and the speed action of an enzyme is said to be its ‘turnover number’. The turnover number is the number of substrate molecules which one molecule of  the enzyme can turn into products per minute of a product ¹. The turnover numbers of an enzyme can vary from 100 to many millions. For most enzymes the turnover number is several thousand. The speed of which some enzymes can work maybe shown by pepsin, for example 30g (1oz) of pure pepsin would be capable of digesting nearly 2 tonnes of egg white in a few hours ².

The fastest known enzyme is called ‘catalase’ and has a turnover number of 6 million. It is found in various tissues where it speeds up the decomposition of hydrogen peroxide (H₂O₂) and converts it into water and oxygen. The enzymes are not destroyed in their reactions and so can be used again and can work in either direction. This means that large amounts of the product are formed.

Enzymes can also convert the product back into the substrate ¹, as shown below:

enzyme + substrate   ↔  enzyme- substrate complex   ↔   enzyme + products

   [ E ]  +  [ S ] ↔                   [  ES  ]             ↔        [ E ] +  [ P ]  

The enzyme is unchanged by the reaction it catalyses.

Enzymes are all proteins and as such share the properties of proteins:

·        Enzymes are affected by heat. Up until about 40^oC the rate increases smoothly at about double the rate of reaction for every ten degrees rise ¹. Above this temperature the rate of reaction begins to decrease because at high temperatures the enzyme denatures. Due to this fact, very few cells can tolerate temperatures over 40^oC – the optimum temperature.

·        The pH of an enzyme’s surroundings also affects its efficiency. Most enzymes function best around pH7 because excessive acidity or alkalinity makes them inactive. However, each enzyme has its own pH range, for example digestive enzymes prefer either acidic or alkaline surroundings.

·        Enzymes are also specific in their reactions to substrates. Usually only certain enzymes will catalyse certain reactions. The degree of specificity varies from enzyme to enzyme, for example digestive enzymes will work on a wide variety of reactions and substrates. Specific reactions can be illustrated by a ‘lock-and-key’ diagram which illustrates the fact the only on type of substrate will fit the enzyme, like putting a key into a lock, as shown below:

Diagram to show the reaction of specific enzymes ²

Effect of concentration:

Generally, the more enzymes there are to catalyse a reaction, the quicker the reaction will proceed. This is because there is more chance of enzyme molecules randomly colliding and reacting with the substrate.

The reverse is also true ; if you increase the concentration of substrate keeping the amount of enzyme fixed, then the rate of reaction will increase proportionately.

This trend continues until there are more substrates than there are enzymes, (or vice-versa) when the reaction will be going at a maximum rate or speed. After this the rate ‘levels off’, and no matter how much more substrate you add, you won’t get any further rate increase.

Therefore if the concentration of substrate is increased using a fixed amount of enzyme, then the results would look like those in the graph below:

2.  Aim of the Investigation

            The aim of this investigation is to look at the effects of different concentrations of hydrogen peroxide on catalase enzyme (found in yeast) and the difference in the rates of reaction for the different reactions.

I designed a pilot study to test my hypothesis.

3.  Hypothesis

I predict that the higher the concentration of substrate (H₂O₂ ), the faster the rate of reaction will be with the catalase and I also predict that if you double the concentration of H₂O₂, you will double the initial rate of reaction.

I expect this hypothesis to be true because at high concentrations of H₂O₂ there will be more substrate molecules present in the solution. This means that there will be more collisions per second between the catalase enzymes and the molecules of hydrogen peroxide substrate and as a result, the rate of reaction will increase ⁴. The rate of reaction increases because more collisions take place per minute in higher concentrations than at lower concentrations.

 Thus, more hydrogen peroxide is decomposed and more of the oxygen product is given off, so the rate of measurable reaction increases.

Graphs to show the effect of (a) enzyme concentration and (b) substrate concentration on the rate of reaction ⁵

Graph (a) shows that with excess substrate, the initial rate of reaction is directly proportional to the enzyme concentration so that if you double the concentration, the initial rate of reaction will also double (at constant temperature and pH). Graph (b) shows that with constant enzyme concentration and a relatively low substrate concentration, the overall rate of reaction is proportional to the substrate concentration (at constant temperature and pH) and that with increasing substrate concentration, the rate approaches a maximum. The same principle is used for enzyme concentration and will produce a similar graph.

I predict that the actual results from my investigation will produce similar graphs for initial rate of reaction and enzyme concentration as shown in graph ‘b’ above.

4.  Independent Variable

My independent variable will be the concentration of the H₂O₂ solution.

H₂O₂  concentartion will be the only input variable because I will keep all the other variables constant. I shall use six different percentage concentrations of H₂O₂  solution at 1%, 2%, 4%, 6%, 8 % and 10%.

I will use a 0% solution comprising water only for my control to adjust my results if any oxygen is given off from the hydrogen peroxide naturally decomposing.

My source of enzyme (catalase) could be any living tissue as all cells contain this enzyme, but for ease of use I have decided to use a standard yeast solution.

5.  Dependent Variable

            This will be the volume of oxygen produced from the reaction between the catalase and the hydrogen peroxide over 30 seconds.

Later I can convert this figure to a volume per minute by doubling.

This will be the only output variable and will be measured in cm³  min^-1. (“centimetre cubed per minute”)

6.  Control Variables

1.      Temperature – As the temperature increases so does enzyme activity. However, this is only true up to around 40^oC. Temperatures higher than 40^oC will cause the enzymes to start to denature. By about 45^oC the enzymes will have passed their ‘optimum’ and will have been denatured. The temperature must be kept constant otherwise the activity of the enzymes will fluctuate and so this will effect the volume of oxygen produced. I will use room temperature and make sure that all of my results are taken within the same hour. This will mean that the temperature should not noticeably fluctuate.

2.      Volume of Hydrogen Peroxide – The volume of hydrogen peroxide must be the same for each experiment because if the volumes are different it will have an effect on the rate of reaction.. I will keep the volume of hydrogen peroxide at 1cm³.

3.      Apparatus -  I will try to keep the apparatus the same between each lesson – this will avoid any problems with different makes of glassware, stopwatches, measuring cylinders etc. which could introduce inaccuracy.

4.      Start of Timing – I will start the stopwatch as I add the hydrogen peroxide to the catalase to ensure that all of the results will be in the same time frame. If the stopwatch is started before the hydrogen peroxide reaches the catalase then there will be a time of no reaction and so the rest of the results will be unreliable because their timing will be wrong. Similarly, if the stopwatch is started after the reaction has started to take place then, the results will be unreliable because they will correspond with the wrong time frame.

5.      Concentration of the Catalase in Yeast Solution – In a high concentration of yeast there will be more catalase enzyme molecules than in a lower concentration of yeast. This means that more enzymes will be able to work at the same time to break down the hydrogen peroxide, and so reaction time will decrease as the enzyme concentration increases. Therefore, I will keep the concentration of the catalase in the initial yeast solution at 5% .

7.  Pilot Method

Diagram to show the set-up of the preliminary experiment

for the pilot method

            This pilot method is designed to help see if my actual method will work and where I can make improvements so that the actual method will be more accurate and give more reliable results. I used the following pilot method:

1.      Collect and set up the apparatus as illustrated in the diagram above. Wear goggles when syringing.

2.      Remove the bung from the test tube and place 1cm³ of 10% catalase (yeast) solution into the boiling tube using the syringe.

3.      Fill the beaker with water. Place the end of the delivery tube into the water and make sure that the end of the delivery tube is completely submerged under the water.

4.      Next, take the 10cm³ measuring test tube and fill it to the top with water. Place your thumb over the end of the measuring test tube so that no water will escape. Turn the tube upside down and place it over the end of the delivery tube. Make sure that the end of the measuring test tube is underwater before you take your thumb away otherwise the water will escape from the measuring tube.

5.      Now take the other syringe and fill it with 1cm³ of 10% hydrogen peroxide. Collect the stopwatch.

6.      Add the hydrogen peroxide and immediately replace the bung. Start the stopwatch as soon as the hydrogen peroxide is added.

7.      Record the volume of oxygen produced by reading it off of the measuring test tube for one minute and collect at least three readings. The experiment must be repeated three times to make sure that you get accurate results.

8.      Now repeat the experiment using 0% (water), 2%, 4%, 6% and 8% concentrations of H₂O₂. Dilute the H₂O₂ with water. For example, to make 8% concentration, dilute 0.8cm³ of H₂O₂ with 0.2cm³ of water. Repeat the above but with different volumes of water and H₂O₂ for the other solutions.

Pilot Study Results

The following table shows the volume of oxygen produced after one minute for different concentrations of H₂O₂:

These results show that my method should work. Most of the results are reasonably consistent. However, I can improve the reliability of my results by changing some of my method.

Pilot Method Improvements

There are a few improvements which could be made to my pilot method including:

·        Instead of an instantaneous rate of reaction I shall record the complete reaction until there is no more change in the volume of oxygen produced.

·        I shall also take the recordings every 30 seconds so that the reaction may be followed more closely.

·        I shall also use  more precise syringes which go up to 1 cm³ and so my results will be much more accurate.

·        I shall also make sure that no residue water or solution is left inside the boiling tube.

·        I shall use a syringe already inserted into the bung to add the hydrogen peroxide. This way no oxygen will escape because I will not have to replace the bung after adding the hydrogen peroxide.

8.  Actual Method

Diagram to show the set-up of the experiment for the actual method

            The method and the diagram above have been changed according to the improvements made to my pilot method. I used the following actual method:

1.      Collect and set up the apparatus as illustrated in the diagram above.

2.      Remove the bung from the test tube and place 1cm³ of catalase (yeast) solution into the boiling tube using the 1cm³ syringe.

3.      Replace the bung and fill the beaker with water. Place the end of the delivery tube into the water and make sure that the end of the delivery tube is completely submerged in the water.

4.      Next, take the 10cm³ measuring cylinder and fill it to the top with water. Place your thumb over the end of the measuring cylinder so that no water will escape. Turn the tube upside down and place it over the end of the delivery tube. Make sure that the end of the measuring test tube is underwater before you take your thumb away otherwise the water will escape from the measuring tube.

5.      Now take the syringe out of the bung and fill it with 1cm³ of hydrogen peroxide. Make sure that the hydrogen peroxide is at 10% concentration. Collect the stopwatch. Add the hydrogen peroxide and at the same time start the stopwatch.

6.      Record the volume of oxygen produced by reading it from the measuring test tube for every 30 seconds until the volume of oxygen remains constant for three consecutive readings. Repeat the experiment at least three times to make sure that you get accurate and reliable results.

7.      Wash out the test tube twice to make sure that all of the previous reactants are gone. Then shake the test tube to remove all of the water. Wash out the syringe to ensure that the concentrations are not mixed.

8.      Now repeat the experiment using 0% (water), 2%, 4%, 6%, 8% and 10% concentrations of H₂O₂. Dilute the H₂O₂ with water. For example, to make 8% concentration, dilute 0.8cm³ of H₂O₂ with 0.2cm³ of water. Repeat the above but with different volumes of water and H₂O₂ for the other solutions.

9.      Make sure that each experiment is repeated three times to make the results reliable.

10. When repeating the experiment with diluted concentrations make that you gently swirl the test tube with the solution inside to make sure that you thoroughly dilute the H₂O₂.

Safety

Make sure that goggles and laboratory coats are worn whenever syringing. Make sure that a damp cloth is nearby to mop up any spilt hydrogen peroxide.

Apparatus

·        1 ´ Test tube rack.

·        1 ´ Stopwatch

·        1 ´ Boiling tube

·        1 ´ Beaker

·        1 ´ ‘S’ shape delivery tube

·        1 ´ Bung with inserted needle

·        2 ´ Syringe

·        20cm³ of hydrogen peroxide at 10% concentration

·        20cm³ of catalase (yeast) solution at 5% concentration

·        1 ´ Test tube with cm³ divisions of at least 0.2cm³

WHAT TO DO NEXT:

Now you have read this plan you need to carry out the practical.

Your teacher will explain exactly how to go about this, but you can use the check-list below to keep a track of your progress.

o       Carry out the pilot method to familiarise yourself with the apparatus.

o       Carry out the main practical and record all results.

o       Draw up a ‘Perfect Table’ of results.

o       Construct a ‘Perfect Graph’ and Analyse the results in detail.

o       Evaluate the method – looking for errors and improvements.

IF YOU GET STUCK:

Speak to your teacher or visit www.skinnersbiology.co.uk

FINALLY:

Don’t miss your deadlines...

Good Luck!