Emil Fisher in 1894, stated that ‘that this was because both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. This is often referred to as "the lock and key" model.
ref. 1.2
When substrate binds to the active site of an enzyme, we commonly refer to it as substrate-enzyme complex. Referring to figure 1.3 activation energy changes when using enzyme and without using it. You can clearly see that activation energy without energy is much higher, than activation energy when enzyme is used. Higher activation energy means that there must be higher energy input to start the reaction. As you can see below, enzymes decrease that amount of energy, so the rate of reaction will be higher using smaller amount of energy.
Figure 1.3
There are many factors affecting the enzyme activity, such as:
- Temperature: According to kinetic theory, the higher the temperature the higher the kinetic energy of the molecules (substrates effectively), so they will vibrate faster. This increases the possibility of the collision between enzyme and substrate and forming of enzyme-substrate complex. Also the energy of the system will be higher, so more molecules will overcome activation energy, so more molecules will react during successful collision.
Ref. 1.3
'The kinetic energy of particles follows the Maxwell-Boltzmann distribution. An increase in temperature not only increases the average speed of the reactant particles and the number of collisions, but also the fraction of particles having kinetic energy higher than the activation energy. Thus, the effective collision frequency increases. '
There is a temperature at which the enzyme works the best (the highest rate of reaction), this is called the optimum temperature. For most of the enzymes this temperature is equal to 37 degrees celsius, as this is the average temperature of the human body, which is why the enzymes work best at this temperature.
However higher temperature than this, the enzymes are said to become denaturated. This means that the shape of the active site is being permanently changed, so the substrate can no longer fit into it. This is caused that the enzymes are vibrating so fast that the internal structure is being changed, due to some bonds may break because of the vibrations, and the consequence of this is that the active site is changed.
-
pH: Different enzymes work best at different concentrations of hydrogen (H+) ions (pH). The charge on the Hydrogen or OH- (hydroxide) ions can affect the charges of side chains of active sites of an enzyme. This will cause the hydrogen bonding that holds enzyme together to be affected, changing the active site of an enzyme, as tertiary structure will be changed (3D representation of protein). Changes in active site of an enzyme will cause that the substrate will no longer fit into the enzyme, so the enzyme becomes useless- they are being denatured.
In this case, too high or too low pH can cause side chains to be so much changed, that the active site’s shape will change, causing the substrate not being able to fit into it. This causes disturbance in the 'lock and key' theory, as the changed shape of the active site of an enzyme will not accept the complimentary substrate, so no enzyme-substrate model can be formed. Picture below shows the effect of pH on enzyme.
fig. 1.4
- Inhibition: If there is a substance that can also bind to the active site of the enzyme, the rate of reaction may decrease, because there will be competition between inhibitor and substrate. This is an additional factor affecting enzyme activity. However in this experiment there will be no inhibition between any substrates, as only one substrates will be present in the solution with the enzymes.
- Concentration of substrates: The more substrates the higher the rate of reaction, as there will be available substrates to bind to active site. However at some point there will be a point at which all of the active sites will be occupied and therefore they will not catalyse reaction.
In this practical investigation I am going to use enzyme called lipase. Lipase is a water soluble enzyme, that catalyses reaction of hydrolysis of ester bonds in lipids. Below is the picture of the mechanism of the lipid breaking down by lipase:
fig. 1.5
As you can see above the molecule of glycerol reacts with water, in presence of lipase, to break it down to 1 molecule of glycerol and three molecules of fatty acids
The substance that aids the above reaction is the bile salts. Bile salts are steroids (‘a class of molecules that contain a similar chemical core. The natural function of steroids is as hormonal messengers in the body. Synthetic versions are administered as drugs because of their ability to reduce inflammation'; ref. 1.3) that show detergent properties. A bile salt is a substance that is the product of the oxidation of cholesterol by the liver. This substance is stored in the gall bladder. Bile salts have many important functions inside the body, such as elimination of cholesterol from the body.
However the most important function for us is that the bile salts emulsify the lipids. Emulsify means that the bile salt, break down the lipids molecule into smaller molecules. This gives advantage for the lipase, as the surface area or the substrate is higher, which causes that the concentration of substrates, one of the factors affecting rate of reaction, will be higher. As I have said before the more the substrates the rate of reaction will be higher up to some point at which all of the active sites will be already occupied, so there will be no enzyme reaction. Bile salts are used to emulsify lipids from food that pass through the intestine, digest fat, which enables absorption of substances across the intestinal walls. Most common bile salts are cholate and deoxyholate.
There also enzymes present in the bile, the most common and important one is Sodium Hydrogen Carbonate (NaHCO3). This enzyme neutralises acid as it enters duodenum (first part of small intestine) from stomach to produce a neutral pH of 7, at which enzymes work best. There is also another important fact that this enzyme is very helpful. This is because; the small intestine is not adapted to cope with low pH of the substances that come from stomach. Such low pH (1 or 2) could damage the lining of the small intestine, including perforating it (punching the wall, so the substance can get outside the small intestine)
The idea of this experiment is to break those lipid molecules down. As I have stated before I will use lipase to do so. Lipase is the enzyme that's active site is complimentary to the fat molecules (substrates), which is why I am going to use it. Also the bile salts will prevent the clumping of fat molecules together as it will emulsify them, which means that it will break them down into smaller molecules. Therefore the concentration of the substrates will be higher which has a direct impact on the rate of the reaction of hydrolysis of the lipids. However there is a problem in this experiment, as the lipid molecule is broken down; the fatty acids are being broke of. As they have ability to donate protons of H+ (Bronsted-Lowry definition of acid) the pH of the solution will decrease, making it more acidic. This makes a problem as the enzymes may become denaturated in low pH, which will cause stop in reaction of hydrolysis as the substrates will no longer fit into the changed shape of active site of an enzyme. The body itself can cope with this applying the substances called buffers to the region in which fats are being broken down.
'Buffer solutions are solutions that resist change in Hydronium ion and the hydroxide ion concentration (and consequently pH) upon addition of small amounts of acid or base, or upon dilution.'
HA(aq) + H2O(l) → H3O+(aq) + A−(aq)
Ref. 1.4
The hydroxide group (OH-) is consumed by the hydroxide ions (H+) and opposite. This helps maintain homeostasis ('that regulates its internal environment so as to maintain a stable, constant condition' ref. 1.5) of the organism.
However in this experiment I cannot use any buffers. This is because there will be a pH change noted. If I was going to use a buffer solution the pH change will not be noticeable, as the buffer will cancel out the change in pH, which means that I can't investigate the aim.
Safety:
Safety is very important issue; this is because working in clean safe environment will ensure that there will be no damage to the experiment or any harm to yourself and the students around you.
In this experiment I will use lipase which in contact with skin may cause irritation. Also ingestion of the bile which I am going to be using can cause irritation to the digestive system and vomiting effective. This is why precautions must be kept using these two chemicals.
There will also be a lot of glass ware around me, which is why awareness that breaking it may cause damage to yourself and others around you. Broken glass may cut the skin and even blood vessels which is dangerous.
There are many electrical points around the laboratory, which may cause electrocution, when the water from e.g. water bath will be spilled on them. Voltage in the sockets is very high and may be fatal to person. This is why the water bath tank should be kept some distance over any of the electrical point.
Contamination of the chemicals can be a possible threat to the experiment. That is why the precaution should be used, that all of the chemicals are separated by some distance.
Also lab-coat should be worn at all times as the chemicals may stain clothes. Goggles are also important as contact of bile salts or enzyme with eyes may be harmful. Any such event should be immediately reported to teacher and eye washed out with plenty of cold-water.
Prediction
Based on the scientific research and the preliminary work I can draw following predictions:
- The higher the concentration of the bile salts the higher the pH's change: This is because, higher concentration of bile salts will emulsify more lipid molecules, so more substrates will be available to form enzyme-substrate complex with the lipase enzyme. This means that there will be higher concentration of substrates available to bind to the active site of the enzyme and to be broken down. The more lipid molecules broken down the more fatty acid triplets produced, so the pH will be changing more noticeably.
However there will be a point at which the pH will be decreasing over small rate. This is because at this point the pH of the solution will drop to the level, at which the pH of the enzyme's natural environment will be so different that the enzyme will not work at its highest rate- it will start becoming denaturated. At this point there will be less hydrolysis reactions, less fatty acid triplets produced, and hence the pH will be dropping at slower rate.
Variables:
Dependent variable is the variables that will automatically change when the independent variable is changed. In this experiment the pH change will be the dependent variable. This is because as I have mentioned before the pH change is directly related to the concentration of the bile salts. The pH change will be different when I am going to use different concentrations of the bile salts (the higher the concentration of bile salts the bigger the pH change). This variable will be controlled by a pH probe, connected to palm top. This pH probe measures pH changes with accuracy to 2 decimal places
Independent variable is the variable that will be changed for a purpose by me, in order to investigate relationship. The independent variable in this experiment will be the concentration of the bile salts. This is because I will vary the concentrations 5 times in order to investigate the relationship between the concentrations of bile salts and the pH change of the solution
Concentrations of bile salts which I am going to use are: 0%; 25%; 50%; 75% and 100%.
Fair testing:
In order to obtain reliable data I will have to keep some apparatus and method of use same, as different apparatus may be different calibrated, and using different methods may give different accuracy (refer to appendix 2 for detailed information). These fair tests are listed below:
- Volume of the lipase: As I have stated in the scientific background, the lipase catalyses the reaction of breaking down of fats. This means that the higher the volume of the enzyme used the more reactions there will be (up to some point at which it will level off, due to not enough amount of substrates to bind to the active site of an enzyme). This is why I have to keep the volume of the lipase constant. I have decided to use 2ml each repeat, because using this quantity in the preliminary work (Appendix 2) I was able to obtain reliable results (pH drop). I will use the 10ml measuring cylinder to measure out this quantity, because it is reliable piece of apparatus.
-
Temperature of the water bath: As I have researched the temperature have the direct impact on the rate of reaction of hydrolysis of the lipids. This is because higher temperature means more kinetic energy per molecule, which makes them vibrate faster and the chance of successful collision is higher. Also more molecules will overcome the activation energy, which combined with faster vibrations increase the rate of reaction. The water bath will be used instead of ordinary flame, because it takes more energy to heat a kilogram of water by 1 degree Celsius (4.2 KJmol-1) than air (about 1 KJmol-1), which means that the water has got higher specific heat capacity than air. The water bath is easily controlled as it can be adjusted to some temperature. Also to make sure that the temperature is right I will put a thermometer into the water bath.
- Same pH change meter: I will use the same pH meter, because different pH probes may be calibrated differently, which would show a variation of the results. I simply will use the pH probe which I have been given every repeat, and after repeat, clean the end of it (probe) with excess of distilled water (neutral pH) and the solution inside the container of the probe). Then leave it for about 5 minutes on the tissue paper in order to dry, so there will be no contamination by another substance.
- Type of food source: Preliminary work has shown that the cream is the most suitable food source for this experiment, which is why I am going to use it. I will have to use same cream at all time, as different creams may have different amount of the fats in them. After collecting apparatus I will use small beaker to pour some of the cream, which then will be used in the actual experiment.
- Volume of the food source: As I have stated in the scientific background, concentration of the substrates have direct relationship with the rate of the reaction. The more substrates can bind to the active site of an enzyme. This means that the higher the volume of the substrates added the more reactions there will be (up to some point at which it will level off, due to not enough amount of the active sites of an enzyme to bind to). This is why I have to keep the volume of the food source constant. I have decided to use 2ml each repeat, because using this quantity in the preliminary work (Appendix 2) I was able to obtain reliable results (pH drop). I will use the 10ml measuring cylinder to measure out this quantity, because it is precise piece of apparatus.
- Keep the temperature of the water bath same- at 40 degrees Celsius. I have to keep the temperature of the water bath the same, as the different temperature have direct impact on the rate of the reaction, as i have told before in the theoretical knowledge. The higher the temperature the higher the rate of reaction, as the particles will have higher kinetic energy, which will make them vibrate faster so the chance of possible collision is higher.
Apparatus list
Method:
- Collect all of the apparatus from the list above
- Clean the desk and the workplace
- Wear lab coat and goggles (safety issue)
- Prepare the results table for the data which is going to be implemented
- Prepare the different concentrations of bile salts (0%; 25%; 50%; 75%; 100%) according to the bottom table of the appendix 1
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Set up water bath to be 40oC and measure its temperature using thermometer
- Pour into each of first set of 3 test tubes amount of chemicals as shown in the table. Do not add cream yet.
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Put into the water bath the boiling tubes and give them 5 minutes to reach the temperature of the water bath. At this point using 3 measuring cylinders pour 5cm3 to each of the measuring cylinders
- Measure the pH of the solution in the water bath and record it in table of results
- Simultaneously pour the cream into boiling tube and start the stop-watch of boiling tube 1. After one minute pass pour cream into boiling tube 2. After one minute pass pour cream into boiling tube 3 (this will allow taking measurement every minute)
- Every minute measure the pH of the solution of test tubes in order 1 then 2 then 3. Record the data into table
- Repeat steps 7 to 11 4 more times using different concentration of bile salt every repeat (according to appendix 1)
- Clean the surface of the desk and wash all of the apparatus
- Process the results
Analysis
The shape of the graphs which I have drawn based on my results I am happy to say that the predictions which I have made were correct. Increasing the concentration of the bile salts shows the biggest pH drop. This means that using most concentrated bile salts, causes biggest increase of concentration of the hydrogen ions, which means that the mixture becomes more acidic (lower pH). This is caused by the fact that bile, produced in liver and stored in gall bladder, emulsifies the lipids. Emulsification means that the bigger molecules of lipids, which tend to “clump” together, are being broken down into smaller groups or even individual molecules. Then enzyme lipase causes them to be broken down into smaller one molecule of glycerol and three fatty acids (hydrolysis reaction). This increases the surface area of the substrates, so it is easier for the enzyme lipase to work at higher rate, as there are more substrates to bind to enzyme's active site, which is the site of the reaction in enzyme. Breaking down the molecule of lipid releases 3 fatty acids, which lower down the pH of the mixture, as they are acidic. This is the reason why the pH of the mixture falls during course of reaction. Based on this knowledge I am able to explain the pH drop of mixture, assuming that the only way to produce hydrogen ions is the by-product of the hydrolysis of lipids reaction.
There is exponential relationship in my graphs, which means that the rate of the reaction (gradient of graph) is constantly changing. This is caused by the ration of enzymes to the substrates. As the course of reaction proceeds, there are more substrates in the mixture than the enzymes. This means that there are free substrates in the mixture, because all of the active sites of the enzymes are already being occupied by the substrates. This means that there are no free enzymes to break down the lipid molecules. The opposite of this may also be happening. There are more enzymes that there are substrates in the solution. This is because more substrates are being broken down; so many active sites of enzymes are not catalysing reaction as there are no free substrates in the solution to bind to the active site of an enzyme. I think that this is better explanation of the process of why the rate is changing.
The third explanation of the decreasing rate of reaction is that the pH drop at some point is so low that the enzymes are starting to become denaturated. Denaturation is occurring when due to changes in the side chains of the proteins (especially amino acids as they are zwitter ion-they consist of both positive and negative charge) as they are being affected the most. The effect of hydrogen bonds on these side chains affects the shape of the enzyme's active site. This change in shape causes that the substrate does not fit any more to the complimentary active site of an enzyme, so the hydrolysis reaction of lipids can not take place. This could be the reason why the rate of breaking down of the lipid molecules was decreasing.
Even though I am happy with this experiment I have noted some point on my graph and in the table of results that I consider to be anomalous results. These are results that are random errors, which occurred not due to wrong procedure, but by the fluctuation of apparatus or by a human error. For example at particular time I had to quickly record the pH and make sure I have done it in good time. This could cause that the pH probe did not give me the actual pH reading of the solution- it given me pH at particular time. I would have to wait some time in order to pH probe stabilise its reading. This could be the cause of these errors. Also even bigger problem that I have noted is that the top of the boiling tube was too narrow for the entire pH probe to fit in. Therefore I had to keep test tube at an angle in order to the bottom part, a measuring bulb, is covered by the mixture. However I will talk more detail about the possible sources of error in the evaluation section.
I have decided to process my graph data in the way to get to know the value of the initial rate of reaction values. I have done that by drawing tangents into all of the lines on the “Graph of pH against the time” and then calculating it by change in value of y-axis divided by change in value of x-axis. I have done this in order to support the theory that increasing the concentration of the bile salt has a direct impact on the rate of the reaction. The beginning of the reaction will give me the best results, as there will be same amount of enzymes no matter on which experiment (as volume of lipase has been kept constant) and there will be difference in the concentration of substrates (as the concentration of the bile salts has been different for each experiment). Below are the results which I was able to come up with:
Evaluation
I am happy with the experiment which I have conducted. I think that I have made effort conducting this, which had benefited me with the reliable results. I have conducted is safely. There are however some possible improvements which I could have make. This is because I have obtained some anomalous results. These results were a little bit off the line of the graph which I have drawn which means that I had to draw a line of best fit. These results could have been made because of inaccuracies of the equipment which I have been using. Another way of conducting random errors was the biggest limitation. This is because the boiling tube was too narrow so the pH probe could not fit. This means that the bulb which actually measures the pH change could not be covered with solution. This is why I had to keep the boiling tube at an angle, so the solution is covering the bulb. This could have been the source of these errors, as the bulb could have not been covered fully. Even with this couple of limitations I was able to obtain data which have supported my predictions.
However if I could do that experiment again I would make following improvements to the apparatus which I would use:
- Wider boiling tubes: this is because the ones that I have been using in this experiment were to narrow, so the pH probe couldn't reach the bottom of the boiling tube. This made me keep the tube at an angle, which I think was the biggest source of the error in this experiment. If I could use wider boiling tubes the error could have been eradicated. This would increase the reliability of my experiment
- More accurate pH probe: I would like to use pH probe that is controlled by the palm top. This is because the timing of the measurements is more reliable and accurate. Also the computer will diagnose better the pH, so the fluctuation of the values will not be noted any more. Also such probe doesn't need to be calibrated each repeat, which could be the source of error. This piece of apparatus would increase the reliability of my experiment.
- Swap measuring cylinders for glass pipettes. This is because there was some solution inside the measuring cylinder after I poured it into boiling tubes- this liquid has been trapped inside the cylinder. The glass pipette is more accurate piece of apparatus as it measures the volume more precisely. Its bigger scale allows better judging what is the exact amount of liquid poured into the tube. Also the glass pipette consists of sucking system which allows eradicating the problem of trapping of the substance, as it can be blown out of it. This is important, as the amount of the substance will be the same each repeat (as even drop of lipase enzyme consist of many enzymes to carry out hydrolysis reaction). This will also improve accuracy and reliability of the experiment
- Mixing of the substances would be another introduction to improved method. This is because I have noted that there was separation between layers of different substances. Stirring it would be a good idea, as there could be some substrates in the bottom layer, while free enzymes could be on the top layer.
- Take repeats every 1 minute. This would increase the reliability of my experiment, as there would be more points on the graph, so maybe the line of best fit wouldn’t have to be drawn.
- Use more concentrations of the bile salts. I would like to change concentrations in 10% intervals. This would allow me to compare accurately the best bile salt concentration for the action of lipase enzyme to hydrolyse lipids. This would increase the strength of the conclusion drawn, and determine best concentration for emulsification.
Above information's are suggestions of improving the experiment. However the results obtained in this experiment are reliable themselves, as they are matching my prediction based on the scientific research. Also they allow drawing conclusions which are supported by both science and predictions.
References:
“Advanced Biology for You”, by Gareth Williams, publisher: Nelson Thornes
Fig.1.1:
Fig. 1.2
Fig. 1.3
Fig. 1.4:
Fig.1.5:
Fig. 1.2a: the graph of preliminary results found in the ‘Appendix 2’
Ref. 1.1
Ref. 1.2
Ref. 1.2 http://en.wikipedia.org/wiki/Collision_theory
Ref. 1.3
Ref. 1.4
Ref. 1.5
Ref. 1.6
Ref. 1.7
Ref. 1.7
Ref. 1.8
Ref. 1.9
Ref. 2.0
