Biology Coursework on Enzymes.
Biology Coursework on Enzymes
Name: Brijel Limbachia
Form: 12.92
Teachers: Mr Lotsu and Mrs Stewart
Enzymes in General
I will first of all talk about enzymes in general. This means that I will talk about what enzymes are, how being in certain conditions affects the enzyme itself. I will also talk about how substances can increase the % transmission between the enzyme and the substrate.
What are enzymes?
The catalysis that takes place in organisms is defined as the acceleration of a chemical reaction by some substance which itself undergoes no permanent chemical change. These kinds of catalysts, which are in biochemical reactions, are called enzymes. Enzymes are responsible for almost all of the chemical reactions in living organisms. Without enzymes, these reactions take place at a rate that is far too slow for the pace of metabolism.
An active enzyme could make a certain reaction speed up, but not all living things need all the reactions to be quick all the time. It's more accurate to say that enzymes react with simpler molecules to produce a stable reaction system in which the products of any reaction are made when they are needed, also in the amount that they are needed.
All known enzymes are proteins. Enzymes are high molecular weight compounds made up of chains of amino acids that are linked together by peptide bonds. As you know that enzymes are composed of proteins, these proteins are globular proteins. These globular proteins have a complex tertiary and sometimes quaternary structure, where polypeptides are folded around each other to form a roughly spherical, or globular shape.
Here is a diagram to show the three-dimensional shape of an enzyme:
The green molecules are the substrate in the enzyme (on the left), and the enzyme itself is on the right.
Here is a diagram to show the three-dimensional structure of an enzyme molecule:
The shape of an enzyme molecule is very important, if the molecule is altered in any way, the enzyme can't combine to its substrate, therefore the enzyme will not be able to function any more. The hydrogen bonds and the ionic forces maintain the enzyme itself, and so their function can be affected by the changes in temperature and also changes in pH can affect their function.
Enzymes have several properties; I shall explain them in full below:
Enzymes are specific, this means that each enzyme usually catalyses only one reaction.
Enzymes combine themselves with their substrates so they can form temporary enzyme-substrate complexes.
Enzymes are not changed or used up by the reaction they catalyse; this therefore means that they can be used again and again.
Enzymes work very quickly and each enzyme has its own turnover numbers.
Enzymes are very sensitive to changes in temperature and pH.
Many enzymes need other chemicals called cofactors, in order to function.
The enzyme function can be slowed down or even stopped by inhibitors.
As you know that enzymes are compounds that are made up of chains of amino acids that are linked together by peptide bonds, here is a diagram to show this:
The Specificity of enzymes
Talking about digestive enzymes, these enzymes catalyse only one reaction. Trypsin, for example, can begin the digestion of a wide variety of foods, which are rich in proteins (eggs, pork, chicken, and Soya). But when you look at how the enzyme Trypsin works at the molecular level, you can see that this enzyme is specific. Trypsin cuts an amino acid chain at a point between a lysine and an arginine residue, and nowhere else. Most proteins have these two amino acids next to each other at some points in their polypeptide chain, and so can be partly digested by Trypsin.
My coursework, which is on enzymes, will investigate the variable of pH. Therefore my aim will be:
Aim: I am investing the effect of pH on the activity of the enzyme Trypsin; this is when this enzyme reacts with the substrate Caesin. I am also investing which pH's make the enzyme Trypsin denature.
Preliminary Work
My preliminary plan was:
Preliminary Hypothesis
I predict that as the enzyme Trypsin is in pH 8 (this is the optimum pH for the enzyme Trypsin) the enzyme will be fully active and therefore the % transmission will be high. But further away from pH 8, (more alkaline or more acidic) it will be less active; this therefore means that the % transmission will decrease.
I am expecting that the enzyme Trypsin react the fastest in pH8, this is because from background research it is said that pH8 is the optimum pH for the enzyme Trypsin.
I will explain more about the enzyme Trypsin and its optimum pH in the actual predication/hypothesis.
This is the first materials list and method I came up with before I started the actual preliminary experiment:
Preliminary Materials
Caesin suspension, 0.5% (using skimmed milk powder)
Trypsin solution, 0.5%
Distilled Water
Test tubes and test tube rack
Graduated pipettes or syringes
Glass beakers
Thermometer
Colorimeter
Stopwatch
Preliminary Method
) Set up a water bath using a beaker at room temp.
2) Put 5cm3 of casein suspension in one test tube and 5cm3 of Trypsin in another.
3) Put both of the test tubes in the water bath (beaker of water at room temperature); leave the test tubes in there until it reaches room temperature.
4) Now make a control test tube with 5cm3 of distilled water with the same amount of casein suspension.
5) Now take out and mix together and immediately add the 5cm3 of pH buffer solution, once added the entire solutions shake, and also the stopwatch should be immediately started as soon as all the solutions were added.
6) Observe the contents of the test tube and record your results, place the contents in a colorimeter every 30 seconds. And record the results obtained, in a suitable table, and plot a graph.
7) Repeat this experiment but using a range of pH's. The range starts from 5 and ends at 10. But you have to make sure that the volume, temperature etc. is kept the same in order for it to be a fair test.
Problems that were encountered in the Preliminary experiment:
After carrying out my first preliminary experiment I encountered several problems. Here are the problems that I encountered and the solution of the problem:
The very first problem that I encountered was that my experiment never worked, the reaction never took place. The problem that I found out that I was using the wrong concentrations, I should not have been using 0.5% casein suspension, I found that I should have been using 4% of casein suspension. This was the reason why the experiment never took place.
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This is a preview of the whole essay
Problems that were encountered in the Preliminary experiment:
After carrying out my first preliminary experiment I encountered several problems. Here are the problems that I encountered and the solution of the problem:
The very first problem that I encountered was that my experiment never worked, the reaction never took place. The problem that I found out that I was using the wrong concentrations, I should not have been using 0.5% casein suspension, I found that I should have been using 4% of casein suspension. This was the reason why the experiment never took place.
The next problem that I encountered was that, the very first results that I obtained came out very weird, I found out that the when the reaction took place the contents in the tube went from cloudy in colour to colourless and then cloudy again. I then found out that having a beaker with water, as a water bath was very incorrect this is because I then had several different room temperatures. This therefore could have affected my reaction, and so caused the "weird" results. Therefore I then decided by myself that an actual water bath would solve my solution. So I therefore used a water bath that was set up at 30oC.
The next problem was that the total volume of the solution, would fill up the test tube. This therefore gave me the fear that everything would come out and spill out. So I solved this problem by using a boiling tube by using a boiling tube I had lots more space for the solution and less chance of spilling.
The other problem that I encountered was that because I was using a colorimeter, I had to place approximately 1-2cm3 of my solution that was reacting in covets. This meant that most of my solution would therefore be used up even though the reaction has not finished. So to solve this problem I increased the volumes of the solution that I was using, so instead of using 5cm3 I would use 10cm3.
Because of these problems that occurred in the preliminary experiments, I was able to solve these problems and therefore I changed the materials list and the method, and used the new materials list and method to do my actual practical.
Preliminary Results
PH 8
Time
% Transmission
% Transmission
% Transmission
30
60
90
20
50
80
210
240
270
30
50
70
90
95
90
90
95
95
35
40
65
70
85
90
90
85
85
38
45
48
50
65
75
80
90
90
As you can see from the results above, you can see that the results tells us that the solution clears and then goes cloudy again. This is to do with the solutions I am using; further explanation will be given when the real results are shown.
Actual Experiment:
Here is the new materials list:
Actual Materials list
Caesin suspension, 4% (using skimmed milk powder)- casein was the substrate that was used in the reaction, caesin is found milk, therefore we can say that caesin is a protein.
Trypsin solution, 0.5%- this is the enzyme that is used in the reaction. Trypsin is one of a group of proteases that is secreted by the pancreas, which is in the upper (small) intestine. Trypsin specifically catalyses the splitting of peptide bonds.
Distilled Water- this is used in the control, the control consists of distilled water and caesin suspension.
Test tubes and test tube rack- this is used to put the separate solutions in such as distilled water, Trypsin solution, pH buffer solution, etc.
Graduated pipettes and syringes- the pipettes is used the put the solution that is reacting in the covets so that I can place it in the colorimeter, the syringes is used to get an accurate volume of each solution that I need, e.g. I would need an accurate volume of pH buffer solution etc.
Water bath- I will need a water bath at 30oC, because I am investigating the effect of pH I have to have temperature as a constant so I thought that I would use a water bath, this would keep the temperature constant and therefore I would affect the reaction.
Thermometer- this is used in order to measure the temperature of the individual solutions.
Colorimeter- this is used to measure the percentage transmission of the reacting solution, what I mean by the percentage transmission is that the colorimeter measures the amount of light that can pass through a solution.
Now what the colorimeter does is that it shows at certain time intervals say 30 seconds the amount of light that goes through the solution (from a cloudy colour to a clear). What I mean by cloudy in colour is that, at this stage the molecules in the solution are big molecule that are may be stuck together therefore has a cloudy milky colour, so when the enzyme starts reacting with these "big molecules" it breaks these down into smaller molecules therefore loses the milky cloudy colour and becomes colourless, now the colorimeter shows this by passing light through the reacting solution. This means that when the solution is milky cloudy less light is able to pass through the actual solution. But when the colour of the solution changes from milky cloudy to colourless then more light is able to pass through the solution.
Stopwatch- this is used to measure the time of the reaction that takes place between the enzyme Trypsin and the substrate caesin.
Boiling tubes and boiling tube rack- this is needed so that the total volume of the reacting solution cannot "over flow".
Labels- this is used to distinguish between the solutions that were used, I placed labels on the syringes so that I would not get it mixed up with other syringes, this would help me stop affecting the reaction.
Actual Diagram of the apparatus used:
Actual Method
) Set up a water bath, and then set the temperature at 30oC.
2) Then put 10cm3 of casein suspension in one test tube, put 10cm3 of Trypsin solution in another test tube. Then after that put 10cm3 of the appropriate pH buffer solution into another test tube. Place all three test tubes into water bath that is set at 30oC. Keep the test tubes in there until reached the temperature required.
3) When the three test tubes of solutions are at the right temperature you can then start the reaction by mixing them altogether into a boiling tube, but immediately start the stopwatch when all the solutions are mixed. And at certain intervals you can place an amount of the reacting solution into the colorimeter and record the results into a suitable table. I will record the amount of light that passes through the solution (% transmission) with time in a table.
4) You have to observe the contents of the test-tube when you place them into a colorimeter. The certain time intervals are every 30 seconds.
5) And the last point is that you have to repeat one experiment 3 times, and repeat the whole experiment but with different pH values. When repeating an experiment, it must be ensured that you keep the constants the same such as volume, time, temperature etc. E.g. if 10cm3 of Trypsin is used, then other experiments 10cm3 of Trypsin will be used. And also the temperature is a constant, so in all the experiments the temperature should always be 30oC.
Actual Fair Test
The experiment that I will do will be a fair test due to the following points below:
Volumes- I will keep the volumes constant, so if the amount of Trypsin used is 10cm3 then it will stay the same. Therefore I can ensure that the volumes won't be changed, as it is a constant in the experiment.
Temperature- I will keep the temperature used all the time at 30oC, because if I do change the temperature then it can affect my whole experiment. This is because the change in temperature could increase or decrease the % transmission, therefore by keeping the temperature constant I avoided a problem for my experiment.
Time- the time interval never changed it was always at every 30 seconds that I would take out the reacting solution and place it in the colorimeter. If I did changed the temperature it would have affected the results that I have obtained.
Solutions- I used the right concentration of the solutions each time such as 4% of casein solution and 0.5% of Trypsin solution. If not it would have affected the results that were obtained.
Because I kept these points as my constants my experiment was therefore a fair test.
Safety
I will now explain the common hazards that were involved in the reaction that I did:
I used Trypsin solution (this was prepared for me in solution by the school), now the hazards that were involved were:
Main hazards: irritating to eyes, respiratory system and skin. May cause sensitisation by inhalation and skin contact.
Health effects-Eyes: dust will cause conjunctival irritation.
Health effects-Skin: material will cause irritation. May cause allergic sensitisation.
Health effects-Ingestion: harmful if ingested in large quantities.
Health effects-Inhalation: Exposure to dust may have the following effects: - irritation of nose, throat and respiratory tract. Inhalation may cause pulmonary sensitisation and may cause bronchial spasms in asthma.
Overall Trypsin is harmful/ irritant.
The casein suspension really had no hazards, as it was milk powder.
Now the pH buffers that I used, now these could have been acidic or alkaline, these could have been irritant as well as harmful. All I know is that NaOH and citric acid was used in order to make the pH buffers. (The pH range is from acidic pH5 to alkaline pH10).
Prediction/ Hypothesis
From background research I have found out that the optimum pH for the enzyme Trypsin is pH8. This therefore suggests that the enzyme Trypsin works best in alkaline conditions. Therefore from this account I can say that the enzyme Trypsin will work at its best in alkaline conditions, so this means that it will work at its best in pH's like 8,9,10 etc. This also suggests that the enzyme will not work as good in pH's below 8, meaning in acidic conditions the enzyme will not work or won't work as good.
I have got scientific knowledge to prove my prediction (hypothesis). I will first talk about how pH actually affects the enzyme and substrate interaction.
Here is a graph to show the effect of change in pH on enzyme activity:
As you can see, that in certain pH's the enzymes will work. But this mostly depends on the type of enzyme it is. In my case, the enzyme Trypsin will work at its best in pH 8, it will also work quite good in alkaline pH's. Basically if the pH increases or decreases much beyond this optimum, the ionisation groups at the active site and on the substrate change. At extreme pH (meaning very high pH's like pH10 and over, or very low pH's like at pH1), the bonds, that maintain the tertiary structure- (meaning the active site)-, are disrupted, and the so enzyme is denatured.
So when the enzyme are at extreme pH's, they become denatured this is because free hydrogen ions (H+) or hydroxyl ions (OH-) affect the charges on amino acid residues, distorting the three-dimensional shape and causing an irreversible change in the protein's territory structure.
Enzymes are particularly sensitive to changes in pH because of the great sensitivity of the active site. Even if a slight change in pH is not enough to denature the molecule, it may upset the delicate chemical arrangement at the active site and so stop the enzyme working.
Most organisms have buffer systems that resist changes in pH, and many are able to excrete excess acid or alkali.
The diagram below shows the effect of pH on the enzyme activity:
Now the enzyme Trypsin is one of a group of proteases that is secreted by the pancreas, which is in the upper (small) intestine. Trypsin also specifically catalyses the splitting of peptide bonds in protein molecules. Now because this enzyme is secreted by the pancreas it must therefore work in an alkaline environment, this is supplied by the pancreatic juices. Bicarbonate is secreted to neutralise the hydrochloric acid from the stomach. So therefore this proves why the enzyme Trypsin works best in an alkaline environment, it also shows how this environment is made. Also due to this happening it helps pH8 be the optimum.
This is how pH can affect the activity of the enzyme Trypsin:
It causes changes in the protein structure.
It changes the binding of the substrate to the enzyme.
It changes the properties of the amino acids or the co-factors that are involved in the catalytic activity of the enzyme.
It alters the ionisation of the substrate.
Now pH affects the 3-D structure of all enzymes, each amino acid has a NH2 group and a COOH group. PH is mostly about the concentration of the H+ ions. So this means that at low pH's and high H+ concentration the major form of these groups will be COOH, also called the "Protonated form". At a neutral pH the major form will be COO. At high pH's the major form will be COO and NH2. But the actual pH at which each group becomes ionised really depends on the particular amino acid and also the environment in which the enzyme is found. So we could therefore say for the enzyme Trypsin that in very low pH's (acidic conditions) the 3-D structure will be affected and so the group will be COOH, etc.
Analysis
Results (Time in seconds and at 30oC)
PH5
Time
% Transmission
% Transmission
% Transmission
Average
30
60
90
20
50
80
210
0
0
0
2
2
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
0
PH6
Time
% Transmission
% Transmission
% Transmission
Average
30
60
90
20
50
80
210
240
270
80
84
85
82
82
80
80
79
82
83
82
81
78
80
81
82
80
83
84
82
81
79
80
80
83
84
82
81
79
80
81
82
PH7
Time
% Transmission
% Transmission
% Transmission
Average
30
60
90
20
50
80
210
240
270
70
71
71
75
80
90
90
90
90
71
71
72
75
75
80
90
90
90
73
75
76
79
80
85
90
90
90
71
72
73
76
78
85
90
90
90
PH8
Time
% Transmission
% Transmission
% Transmission
Average
30
60
90
20
50
80
210
240
270
30
50
70
90
95
90
90
95
95
41
50
75
89
95
90
95
95
95
35
50
75
90
95
90
90
95
95
35
50
73
90
95
90
92
95
95
PH9
Time
% Transmission
% Transmission
% Transmission
Average
30
60
90
20
50
80
210
240
270
300
330
94
98
98
98
98
99
99
99
99
99
96
96
98
98
98
98.5
99
99
99
00
96
96
96
98
98
98
98
98
99
99.5
00
95
97
97
98
98
99
99
99
99
00
00
PH10
Time
% Transmission
% Transmission
% Transmission
Average
30
60
90
20
50
80
210
240
270
300
330
94
96
98
98
99
00
00
00
92
96
97
98
98
98
99
99
00
00
95
97
98
98
98
98
99
99
00
94
96
98
98
98
99
99
99
00
00
The results that are in bold black indicate that they are anomalous results.
Explanation of results
From my results you will see many things. The first thing that you will notice is that from pH5 to pH6 you will see from the results that the solution goes clear then dark and then clear again, as from the results table in pH6 are:
Time(s)
Average (% transmission)
30
60
90
20
50
80
210
240
270
80
83
84
82
81
79
80
81
82
As you can see from the results at first the solution start to clear from 30 seconds to 90 seconds the % transmission was from 80 to 84, but after 90 seconds so at 120 seconds the solution goes from 84 down to 82 (this shows that the solution is getting darker). From 120 seconds to 180 it goes down but at 210 seconds we see that the % transmission goes up to 80 this shows that the solution is getting clearer again.
But from pH7 onwards this colour change in the solution doesn't take place. I have got a scientific solution for this. I have two proteins that are reacting, now when they first react you see the solution clearing; this is when the proteins have turned to amino acids, as the enzyme has broken big molecules into smaller ones. After the solution clears we then see the solution getting slightly darker, now this is when the amino acids start to stick together, at this stage we see the solution getting darker. We then see the solution going clear again, this is when the proteins react again and so the amino acids that were stuck together separate.
Here is a diagram to show this happening:
But we only see this colour change in solution in pH's 5 to 6. This is because as I have told you in the past that the enzyme Trypsin works best in an alkaline environment, so in the acidic pH's the enzyme doesn't work as good as the enzyme is in an unfavourable environment. Also from pH7 (neutral) onwards you do not see this happening as the enzyme is in a favourable environment. This proves that part of my prediction was correct, which was that the enzyme Trypsin will not work at its best in an acidic environment.
From the table and from the graph you will see that I have got a few anomalous results. I believe that the main reason for these anomalies is:
Human error- misread the time, got mixed up with the syringes therefore inaccurate experiment took place.
In the graphs I got the following anomalies in pH5, pH6, pH7 and pH8.
From all of the graphs you can see that for all the pH's the results are curves of different kinds. None of the graphs are straight lines.
From the graphs you can see that the higher you go in pH the higher the % transmission becomes.
On the graph of pH 5 you can see that the % transmission is low, this proves that my prediction was correct as I proved that at acidic conditions or pH's the % transmission would be low, as the enzyme Trypsin works best in alkaline conditions. In the graph of pH 5 the error bars are small this shows that I was very accurate during that part of the practical. In the graph of pH5 the % transmission is low because during the reaction the enzyme got denatured, as there was too many H+ ions that affected the enzyme as well as the substrate therefore the solution did not clear completely.
In the graph of pH 6 you can see that the % transmission has become slightly higher than pH5, in that graph you can also see that the error bars are very small again this also shows that I have been very accurate. This graph also proves my prediction correct which was that the enzyme Trypsin would not work as good in acidic pH's. Also in pH6 there were many H+ ions so again the enzyme got denatured, and so the solution didn't clear completely.
In the graph of pH 7 you can see that the % transmission has increased more higher than pH6, in that graph you can also see that the error bars are slightly bigger than in pH6 this shows that I have been less accurate than before, as the error bars have increased. In pH7, which is neutral, the enzyme did not get any hydrogen ions to denature the enzyme, but there were less hydroxyl ions and so the enzyme could not function as well as it could have
In the graph of pH8 you can see that the % transmission has increased again more higher than pH7, in that graph you can also see that the error bars are slightly bigger than in pH6 this shows that I have been less accurate than before, as the error bars have increased. This graph also proves my prediction correct which was that the enzyme Trypsin would work good in alkaline pH's and it has, as the % transmission is higher in an alkaline pH. Now in pH8 the enzyme did not receive any hydrogen ions and so didn't get denatured, but it received hydroxyl ions and so it helped the enzyme function better therefore there was an increase in % transmission and so the solution cleared more.
In the graph of pH9 you can see that the % transmission has increased again more higher than pH8. You can see that the line is less curvy and more straight this shows that the reaction must have taken place very quickly, in that graph you can also see that the error bars are smaller than in pH8 this shows that I have been more accurate than before, as the error bars have decreased. This graph also proves my prediction correct which was that the enzyme Trypsin would work good in alkaline pH's and it has, as the % transmission is higher in an alkaline pH. Now in pH9 the enzyme did not receive any hydrogen ions and so didn't get denatured, but it received hydroxyl ions and so it helped the enzyme function better therefore there was an increase in % transmission and so the solution cleared more.
In the graph of pH10 you can see that the % transmission is the same as the last pH. You can see that the line is less curvy and more straight this shows that the reaction must have taken place very quickly, in that graph you can also see that the error bars are small so it shows that I have been more accurate than before, as the error bars have decreased. This graph also proves my prediction correct which was that the enzyme Trypsin would work good in alkaline pH's and it has, as the % transmission is higher in an alkaline pH. Now in pH10 the enzyme did not receive any hydrogen ions and so didn't get denatured, but it received hydroxyl ions and so it helped the enzyme function better therefore there was an increase in % transmission and so the solution cleared more.
Now I said that in my prediction that from background research the optimum pH for the enzyme Trypsin was pH8, but from the graph and the results in the table we see that the optimum pH is 10, I believe that this took place because I had a constant temperature of 30oC, now the background research said that the optimum pH for the enzyme Trypsin is pH8, this is at body temperature as Trypsin is found in the body. But the Trypsin that I used was a proteolytic enzyme that was extracted from a Bovine pancreas. So this could have been the reason for the change in optimum pH.
As I got a few anomalies it show that I have been fairly accurate in the whole practical.
Evaluation
I believe that overall, meaning after doing the whole experiment I think that I was quite accurate. At first I was very confused as I got odd results and the graphs that I made were not as I expected it to be. But after looking at my results in detail I then understood that my results are correct and fairly accurate. I would still say that my results are fairly accurate after getting a few anomalies. My results do help prove that my prediction was correct overall. My results relate very well with what is happening between the enzyme and pH, e.g. in and acidic environment the enzyme denatures quicker and the "odd" results take place (the solution clears, darkens, and then clears again).
What I expected to get as in the results is what I got, I just didn't understand at first what the results were telling me. As the results proved my prediction correct overall.
The difficulties that I experienced during the whole experiment are:
Using the colorimeter- I didn't know how to use it at first, as I didn't understand what the % transmission meant. I have now learnt from this experiment what a colorimeter is and how to use it. Also what a colorimeter shows you in terms of science.
The time interval- as it was every 30 seconds I had to be very quick. So human error could have taken place, hence the anomalies.
Time- I had very little time, and preparing the whole experiment, meaning getting all the volume right and temperature etc. took time and therefore I had to be quick overall.
I could say that because of these difficulties I got anomalies in the results.
If I were to do this experiment again I would do the following changes:
I would have a control as it helps explain what is taking place between enzyme and pH buffer solutions. Help me to see what I am expecting.
I would also do a test on the enzyme and substrate on its own without temperature etc. affecting it. This also helps me to see what I am expecting.
I would change the time interval to say every minute instead of 30 seconds this can help me avoid anomalies.
I would change the method.
I would have to have more time in order to not rush and so avoid anomalous results.
I would not have 30 degrees Celsius at the constant temperature. This helps get accurate results.
I would change the constants such as volumes of the solutions.