The optimum temperature of the enzyme is the teperature in between the two factors at which the enzyme functions properly. At temperature above the optimum temperature the enzyme becomes denatured. This is when the bonds holding the shape together are broken. The primary structure is retained, however the polypeptide chains unravel and loose their specific shape. Once an enzyme has denatured it is unable to bind with a substarte and has therefore lost its catalytic properties. Denaturation is nearly always irreversiable it can also be cused y changes in pH and salt concentration.
Most enzymes have and optimum pH at which they function most effectively. At extremes of pH values, the enzyme molecule may become denatured. This is due to the changes in pH that affect the ionisation of the side groups in the amino acid residues. This effects the overall shape of the molecule and the efficiency of the enzyme-substrate complex formation decreases the rate of reaction. The diagram below shows a typical graph for the reaction time aginst pH:
Changes in both substrate and enzyme concentration also effect the rate of reaction. An increase in enzyme concentration, increases the number of active sites, therefore more enzymes-substrate complexes can form. This increases the rate of reaction, provided there is an excess of substrate molecules. The rate of reaction depends on the rate of formation of enzyme-substrate complexes. So if the substrate concentration increases, the rate of reaction will increase until there are no more enzymes to bind to. This will result in the rate reaching a maximum velocity and remains constant as the enzymes will prevent an increase in rate. The diagrams below show the effects of enzyme concentration on reaction time and the effect of substrate concentration on reaction time:
For my investigation, I have chosen to investigate the activity of the enzyme Urease. The enzyme Urease is present in many simple organisms. It occurs in many bacteria, several species of yeast and a number of higher plants. Two of the best sources of Urease are jack beans (canavila eniformis), and bacillus pasteui.
Soya beans (and other legume beans) are normally toasted to make them fit for human consumption. This is because Soya contains substances called Trypsin inhibitors that can cause gut upset to people and animals if they were to eat raw Soya beans. These are difficult to test in the laboratory but Soya also contains the enzyme Urease (relatively harmless compared to Trypsin inhibitors). Various studies have shown that Urease is inactivated at the same rate as Trypsin inhibitors by the toasting process. So if the Soya tests low for Urease activity it means they have been toasted properly to remove Trypsin inhibitors.
Urease enzyme occurs throughout the animal and plant kingdom but jack beans seem to be one of the richest natural sources of Urease enzyme. Urease converts urea + water to carbon dioxide + ammonia (NH3). This is quite an important reaction in nitrogen metabolism. Urease is high in plants that are legumes (nitrogen fixers) and whose seeds are rich in protein.
Urease activity in soil (from micro organisms in the soil) is also an important part of the nitrogen cycle in ecology. Urease activity is also an important activity of some important pathogenic bacteria. This is because the ammonia produced by the infecting bacteria is toxic and an irritant.
The enzyme Urease catalyses the hydrolysis of toxic urea into ammonia and carbon dioxide. This can be shown in the balances equation below:
Urea + water carbon dioxide + ammonia
CO(NH2)2 + H2O CO2 + 2NH3
Urea is one of the compounds that make up nitrogenous waste in mammals. Breakdown of access amino acids during deamination produces ammonia. Ammonia is a very toxic and can only be safely excreted if large amounts of water are available to dilute it to safer levels. This is not possible in aquatic or terrestrial or animals such as mammals. These mammals convert the ammonia in the body to urea, which is much less toxic. Urea is a very small molecule and is soluble in water. It diffuses readily across cell membranes and may be found in small amounts in most body fluids.
The production of ammonia raises the pH of the solution and can be detected by using a suitable acid base indicator.
Urease is active over a wide range of pH so any number of indicators can be used provided that the original solution is buffered at the right level to start with.
There are two different types of indicators that I am going to use for this experiment, phenolphthalein and Bromothymol Blue. Phenolphthalein changes from colourless to pink at quite a high pH9. At these pH’s a small amount of ammonia doesn’t affect the pH very much so the transition from clear to pink occurs only very slowly. As a consequence, large variations can occur in determining the first faint pink coloration required for the end-point.
For this reason I am also going to use Bromothymol Blue. This is a better indicator of the two as the colour change, yellow to blue, occurs around pH6 to pH8. The midpoint colour, turquoise, occurs at about pH7.5. At these pH’s a small amount of ammonia will produce a large change in pH so the end point occurs very quickly. This leads to little variation in the timing of the end-point.
A buffer solution is a solution that contains a weak acid and a salt of a weak acid. Depending on the relative concentrations of the weak acid and the salt, the solution will have a characteristic pH value. The term pH refers to the relative concentrations of H+ ions in a solution. The pH is the negative of the logarithm of the molar concentration of H+ ions. A solution is classed as an acid if there is a high concentration of H+, which indicates a low pH value, (below pH7). Similarly a solution is classed as a base if there is a low concentration of H+, which indicate a high pH value, (above pH7). PH7 is neural, neither acid nor base.
Buffer solutions are have an important role in maintaining a particular pH value of a solution, as they resist changes in pH. They do this by neutralising a solution. If more acid is added to a buffer solution, which tends to decrease the pH as by adding an acid to the buffer solution increases the concentration of H+ ions, the excess hydrogen ions are effectively neutralised by the salt of the weak acid. If a base is added to the buffer solution, which will increase the pH as by adding a base to the buffer solution decreases the concentration of H+ ions, the lack of hydrogen ions are neutralised by the weak acid by supplying extra ions.
Acid – base indicator are organic substances that are weak acids themselves. When a reaction is complete, an indicator changes colour to determine the end-point of the titration. Generally, this can be represented by the following equation:
HIn(aq) H+(aq) + In-(aq)
Acid Base
A change in pH causes a shift in equilibrium, which causes a colour change of the acid, or its conjugate base. A dramatic colour change shows the presence of a good indicator, as it is clearer to detect the end-point of a reaction.
Acids and alkalis can be classified as strong or weak depending on the extent to which they form ions when dissolved in water. Strong acids and alkalis are completely in the form of ions in a dilute solution, whereas for weak acids and alkalis, this process is only partially complete. I will make sure that I only use enough of the indicator to give an observable colour change.
Hypothesis
As the enzyme concentration of Urease increases the reaction time of urea broken down into ammonia and carbon dioxide will increase. To prove my hypothesis I will be using the spearman rank correlation test and will be looking for a positive correlation. I will be taking 12 results of each experiment to generate useful, accurate, reliable concordant results and to provide suitable data for analysis of the spearman rank correlation test.
Requirements
Below is a list of all the apparatus and chemicals I will need for my experiments and the reasons for my choices. Before using my apparatus I will make sure that everything is thoroughly cleaned first and rinsed with distilled water to remove any impurities, and dried out thoroughly.
Apparatus:
- Weighing boat – to weigh out solid powder
- Sensitive electronic balance - to weigh solid powder accurately (2dp)
- Glass stirring rod – to mix solution
- Spatula – to transfer solid powders into weighing boat and flasks
- Buchner funnel – for quick and easy to use vacuum filtration
- Suction apparatus - for quick and easy to use vacuum filtration
- Filter paper - for quick and easy to use vacuum filtration
- Access to tap – to create vacuum
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100cm3 volumetric flask &stopper (x2) – to contain standard solutions accurately
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200cm3 volumetric flask &stopper (x2) – to contain standard solutions accurately
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1000cm3 volumetric flask &stopper (x1) – to contain standard solutions accurately
- Funnel – for accurate manipulation of solutions
- Test tubes ( 8 x 12) – reaction vessel
- Test tube rack – to hold test tubes
- Labels – to avoid any confusion with solution and prevent anomalies in my results
- Syringe (x4) – to measure and transfer small amounts of solutions accurately
- Stop clock – to measure the time
Chemicals:
- Distilled water and wash bottle - to rinse out impurities and dilute solutions
- 3% Urease solution – source of enzyme
- 2% urea solution – source of substrate
- pH5 buffer solution – to maintain pH of solution
- pH7 buffer solution – to maintain pH of solution
- pH7.5 buffer solution – – to maintain pH of solution
- Phenolphthalein indicator – to indicate end point of the reaction
- Bromothymol Blue indicator – to indicate end point of the reaction
Risk assessment
Below is a list of all the chemicals I will be using for my experiment and the risks involved with them:
- Urea - toxic
- Ammonia - highly toxic
- pH5 solution - weak alkali
- pH7 buffer solution -
- pH7.5 buffer solution -
- Urease enzyme -
- Phenolphthalein indicator – irritant to eyes, skin and respiratory system.
- Bromothymol Blue indicator -
Safety
Extreme care must be taken when handling all the chemicals mentioned above, as they can be hazardous not only to myself but to other people around me. To ensure the safety and minimise risk from the nature of the hazards exposed to others, and myself I will:
- Make sure that protective clothing i.e. safety glasses; lab coat and protective gloves are worn throughout the experiment.
- Keep hazardous chemicals away from naked flames, as the chemicals I am using are flammable.
- Carry out my experiment in a ventilated fume cupboard as the chemicals I will be using give off irritating vapours. The fume cupboard will ensure that all toxic gases escape successfully.
- Make sure that there are no leading wires from the pipes, the electronic cables from the electronic balance.
- Make sure that water and solutions are kept away from electrical appliances.
- Take care when handling glassware to avoid breakages. If an accident does occur, I must notify my teacher and clear up the debris thoroughly. Any spillages must be cleaned up immediately.
- Make sure all waste chemicals are disposed of safely.
In case of contact with the eyes or skin with any of the chemicals I am using, I must rinse out immediately with plenty of water!
Procedure: Making up the solutions
3% Urease solution
First of all to make my 3% Urease solution, I will place a clean weighing boat on a sensitive electronic balance and re-zero it. I will then transfer the solid powder of Urease active meal using a clean spatula until the reading on the balance reads 3.00 grams. Next, I will transfer the solid into a clean 100cm3 beaker using the spatula. Next, I will measure out 100cm3 of distilled water using a measuring cylinder, making sure that the bottom of the meniscus sits on the 100cm3 graduated line. Using some of the distilled water in the measuring cylinder, I will carefully wash out the weighing boat with distilled adding the washings into the beaker to ensure the entire solid has been transferred. I will then add some more of the water into the beaker, still retaining some water in the measuring cylinder, and stir the solution using a clean glass rod. After stirring, I will filter off the remaining un-dissolved material using vacuum filtration method (see diagram below). Once filtered, transfer all the filtrate solution to a volumetric flask using a funnel. Using the remaining distilled water in the measuring cylinder, wash out the vacuum filteration apparatus and add the washings into the flask. Finally, place a stopper on top of the volumetric flask to prevent any evaporation of the solution and shake the flask vigorously to ensure compete mixing of the solution. I will make sure that my thumb is placed on top of the stopper when shaking to avoid any spillages of the solution. This solution will need to be made fresh.
2% urea solution
First of all to make my 2% Urea solution, I will place a clean weighing boat on a sensitive electronic balance and re-zero it. I will then transfer the solid powder of urea using a clean spatula until the reading on the balance reads 20.00 grams. Next, I will transfer the solid into a clean 1000cm3 beaker using the spatula. Next, I will measure out 1000cm3 of distilled water using a measuring cylinder, making sure that the bottom of the meniscus sits on the 1000cm3 graduated line. Using some of the distilled water in the measuring cylinder, I will carefully wash out the weighing boat with distilled adding the washings into the beaker to ensure the entire solid has been transferred. I will then add some more of the water into the beaker, still retaining some water in the measuring cylinder, and stir the solution using a clean glass rod. Once the entire solid has dissolved, I will transfer all the solution to a 1000cm3 volumetric flask using a funnel. Using the remaining distilled water in the measuring cylinder, wash out the vacuum filteration apparatus and add the washings into the flask. Finally, place a stopper on top of the volumetric flask to prevent any evaporation of the solution and shake the flask vigorously to ensure compete mixing of the solution. I will make sure that my thumb is placed on top of the stopper when shaking to avoid any spillages of the solution.
PH5 buffer solution
First of all to make my pH5 buffer solution, I will place a clean weighing boat on a sensitive electronic balance and re-zero it. I will then transfer the solid powder of urea using a clean spatula until the reading on the balance reads 2.40 grams. Next, I will transfer the solid into a clean 200cm3 beaker using the spatula. Next, I will measure out 200cm3 of distilled water using a measuring cylinder, making sure that the bottom of the meniscus sits on the 200cm3 graduated line. Using some of the distilled water in the measuring cylinder, I will carefully wash out the weighing boat with distilled adding the washings into the beaker to ensure the entire solid has been transferred. I will then add some more of the water into the beaker, still retaining some water in the measuring cylinder, and stir the solution using a clean glass rod. Once the entire solid has dissolved, I will transfer all the solution to a 200cm3 volumetric flask using a funnel. Using the remaining distilled water in the measuring cylinder, wash out the vacuum filteration apparatus and add the washings into the flask. Finally, place a stopper on top of the volumetric flask to prevent any evaporation of the solution and shake the flask vigorously to ensure compete mixing of the solution. I will make sure that my thumb is placed on top of the stopper when shaking to avoid any spillages of the solution.
PH7 buffer solution
First of all to make my pH7 buffer solution, I will place a clean weighing boat on a sensitive electronic balance and re-zero it. I will then transfer the solid powder of urea using a clean spatula until the reading on the balance reads 4.80 grams. Next, I will transfer the solid into a clean 200cm3 beaker using the spatula. Next, I will measure out 200cm3 of distilled water using a measuring cylinder, making sure that the bottom of the meniscus sits on the 200cm3 graduated line. Using some of the distilled water in the measuring cylinder, I will carefully wash out the weighing boat with distilled adding the washings into the beaker to ensure the entire solid has been transferred. I will then add some more of the water into the beaker, still retaining some water in the measuring cylinder, and stir the solution using a clean glass rod. Once the entire solid has dissolved, I will transfer all the solution to a 200cm3 volumetric flask using a funnel. Using the remaining distilled water in the measuring cylinder, wash out the vacuum filteration apparatus and add the washings into the flask. Finally, place a stopper on top of the volumetric flask to prevent any evaporation of the solution and shake the flask vigorously to ensure compete mixing of the solution. I will make sure that my thumb is placed on top of the stopper when shaking to avoid any spillages of the solution.
PH7.5 buffer solution
First of all to make my pH7.5 buffer solution, I will place a clean weighing boat on a sensitive electronic balance and re-zero it. I will then transfer the solid powder of urea using a clean spatula until the reading on the balance reads 0.80 grams. Next, I will transfer the solid into a clean 100cm3 beaker using the spatula. Next, I will measure out 100cm3 of distilled water using a measuring cylinder, making sure that the bottom of the meniscus sits on the 1000cm3 graduated line. Using some of the distilled water in the measuring cylinder, I will carefully wash out the weighing boat with distilled adding the washings into the beaker to ensure the entire solid has been transferred. I will then add some more of the water into the beaker, still retaining some water in the measuring cylinder, and stir the solution using a clean glass rod. Once the entire solid has dissolved, I will transfer all the solution to a 100cm3 volumetric flask using a funnel. Using the remaining distilled water in the measuring cylinder, wash out the vacuum filteration apparatus and add the washings into the flask. Finally, place a stopper on top of the volumetric flask to prevent any evaporation of the solution and shake the flask vigorously to ensure compete mixing of the solution. I will make sure that my thumb is placed on top of the stopper when shaking to avoid any spillages of the solution.
Procedure: Carrying out the experiment
First of all, I will label 8 test tubes 1-8. Next, I will add the required buffer solution, water and urea solution to each of the tubes (refer to table 1 below), using a clean syringe for each of the different solutions. Then, I will add four drops of Phenolphthalein indicator to each test tube. Then I will add the required amount of Urease solution to each of the test tubes (see table 1) and start timing. I will shake the tubes to ensure the contents are mixed thoroughly and then record the time when each tube shows the correct end point colour. I will carry out a rough experiment first of all to indicate the end point, and disregard this result as the first time I will probably over shoot the endpoint. I will then repeat this 12 times so an average can be taken, and to provide suitable data for analysis to produce accurate, reliable, and concordant results. I will then repeat the above procedure using Bromothymol Blue indicator. For this experiment I will set up a controlled test tube by adding 5ml of pH7.5 buffer solution and adding four drops of the indicator. This shows the turquoise mid point colour change that can be used to determine a ‘standard’ end point for the reaction.
Table 1 to show the quantities of reactants needed to add to each test tube:
*use pH5 buffer if using Bromothymol Blue
use pH7 buffer if using Phenolphthalein
Fair test
In order to produce a fair test for my experiment, and keep experimental error to a minimum, to produce accurate, reliable and concordant results the following points must be considered when carrying out the procedure.
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When measuring quantities and taking readings e.g. using measuring cylinders, pipettes, volumetric flask, etc., I will ensure that the value is taken from the bottom of the meniscus level that sits on the graduation line and also that the eyes are positioned at 90o angle to the scale of the apparatus to achieve an accurate and reliable reading.
- When using a thermometer I will make sure that it remains in contact with the temperature of the substance it is measuring at all times during the experiment. This will ensure that any readings are not missed, as the thermometer will not have to continually adjust to the new temperature of a particular environment. I will also make sure that the highest reading on the thermometer scale is also read to prevent any inaccuracy and non-reliable evidence.
- I will make sure that all the chemicals and samples I use for my investigation come from the same source to provide concordant results.
- I will label all solutions possible to avoid any confusion of substances during my investigation, to avoid any uncertainties and anomalous results.
- I will repeat results12 times so an average can be taken, and to provide suitable data for analysis to produce accurate, reliable, and concordant results.
- I will also make sure that all variables are kept constant during the entire investigation to produce reliable results. The variables that I will need to be controlled are as follows
- pH concentration
- temperature
- substrate concentration
Results
Below is a table of results showing the reaction time in minutes, of enzyme concentration using phenolphthalein indicator.
Experiment 1- using phenolphthalein
Below is a table of results showing the reaction time in minutes, of enzyme concentration using Bromothymol blue indicator
Experiment 2 - using Bromothymol blue
Analysis
Below is a table showing the reaction time converted into seconds.
Experiment 1: phenolphthalein
Experiment 2 - using Bromothymol blue
Below is a table showing the average of my results in seconds, the reaction
time in seconds, and the weighted reaction time.
Average = 1 + 2 + 3 + 4 + 5 + 6 + 7 + 8 + 9 + 10 + 11 + 12
12
weighted reaction time = average reaction time (secs) X conversion factor
Experiment 1: phenolphthalein
Experiment 2: Bromothymol blue
