Main Investigation
The focus of this investigation is to investigate the effect of duration of exposure at high temperatures on Trypsin denaturation.
Prediction
As the duration of exposure increases, the time of clarification will increase. However, I predict that although there will be a net increase in the time of clarification, it will be lower at the highest temperatures in comparison to the lowest temperature in the experimental range
Reasons
Duration of exposure
The duration of exposure to a high temperature and its effect on Trypsin denaturation is one of the variables being investigated. Suitable intervals and a range will be chosen to a measure this effect. The degree of denaturation depends on duration of exposure to a particular temperature.
Temperature
Temperature or a range of high temperatures is the other variable to be investigated and has considerable effect on the activity of an enzyme and denaturation. In general, it would be preferable to use enzymes at high temperatures in order to make use of the increased rate of reaction plus the protection it affords against microbial contamination. However, due to the fact that enzymes are globular proteins, high temperatures affect them. Above the optimum temperature, commonly between 40oC and 45oC, many enzymes are denatured. This is the highest temperature at which the rate of reaction is speeded up, without at the same time inactivating the enzyme.
Above the optimum, the enzyme molecules are being progressively destroyed, so they can no longer catalyse the reaction. The process of denaturation is the irreversible destruction of the tertiary structure of the enzyme, changing and permanently destroying the shape of the active site. The substrate can no longer fit into the shape of the active site and so the rate of reaction decreases.
Variables to be investigated
- Duration of exposure
- Suitable range of high temperatures
Variables to keep constant
It is important that other than the factors being investigated, other variables must be kept constant so that they do not influence the way in which the reaction is occurring or affect the experimental variables.
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Temperatures at which Trypsin is exposed to; temperature effects the rate at which an enzyme works. It is important that the temperature at which Trypsin is incubated remains constant throughout the duration of exposure. This is so that the effect of the chosen temperatures on Trypsin denaturation can be deduced.
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Volume and concentration of enzyme and substrate; if the concentration of Trypsin increases, the time of clarification will increase within the 25 minute time period. However, once all the active sites are occupied, the optimum activity of the enzyme is achieved. At this stage, if more product is added, there is no increase in rate of product formation. If there are more substrate molecules than enzyme molecules then the number of active sites is s limiting factor, the concentration of the casein will be decreased with the use of distilled water. To ensure fair testing is maintained, the volume of Trypsin and casein must be kept constant, and so distilled water will be added to the casein to make up the total volume of 5cm3, which will be the same for the Trypsin.
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PH; enzymes are sensitive to pH and most have optimum pH ranges in which they work best. Outside this range, the ph will denature. This is due to the H+ and OH- ions which are responsible for ionising the R groups on amino acids, and as a result, effecting the secondary and tertiary structure of the active site, rendering it inactive. Trypsin’s optimum pH range is between 7.1 and 8.2. As the casein suspension is milk based, I assume its pH range should be neutral or between 7.0 and 8.0, which is appropriate for the activity of Trypsin.
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Time intervals of 5 minutes; duration of exposure is the experimental variable and so a sufficient duration of incubation intervals are needed- 5minute intervals gave sufficient results by an evident change in time reaction rate for the preliminary experiments and so will be used for the main investigation.
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Same methods and apparatus throughout the investigation; to ensure fair testing is maintained.
Justification of procedures
- The enzyme Trypsin will be used in the investigation, as past experiments have showed interesting results between the reactions of this enzyme with casein suspension.
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The diluted casein ratio consisting of 3cm3 casein and 2cm3 of distilled water to make up a total volume of 5cm3 of substrate; this is because it gave me the fastest rate at which the casein suspension cleared, and this time of clarification increased with increased duration of exposure.
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The following temperature ranges of 70, 80 and 90oC; as this is the experimental variable as a range of high temperatures and its effect on Trypsin denaturation is being investigated. In general, it would be preferred to use enzymes at high temperature to make use of the increased rate of reaction.
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Trypsin will be incubated using a manual water bath via the Bunsen burner method as thermostat water baths for considerably high temperatures such as 70,80 and 90oC is not economically feasible due to high degree of evaporation, and in addition this may be dangerous. It is increasingly important that the temperature of Trypsin incubation remains constant throughout the duration of exposure.
- Duration of exposure is the other experimental variable and so I need a sufficient range of 5 settings- 5,10,15,20,25 minutes from which a sufficient change in reaction rate can be determined as seen in the preliminary results.
- All enzyme samples will be cooled down to room temperature after heating; this will enable me to look at the direct effect of the duration of exposure at high temperatures on Trypsin denaturation. However, I am aware that room temperature may fluctuate not only due to a day-to-day basis, but also because of the heat given off by water baths in the laboratory.
- The method used to judge the end-point; white card with a black cross on it will be used to judge any change in casein clarification as opposed to black card as I found this method easier in the preliminary experiments.
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The procedure will be repeated three times in total for each time interval at temperatures 70, 80 and 90oC in order to obtain averages and generate useful and reliable results.
Apparatus
- Casein suspension 4%
- Trypsin solution 0.5%
- Distilled water
- Test tubes and rack
- 5ml syringes
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100cm3 measuring cylinders
- Glass beaker, Bunsen burner
- Thermometers
- White card with black cross
Procedures
- Set up water bath according to availability of appropriate temperatures. If a particular temperature is not available, make up a water bath using test tubes held in a beaker in a beaker of water heated by a Bunsen burner. The temperature can be regulated and kept constant by moving the Bunsen in and out of the tripod from beneath and kept constant by moving the Bunsen in and out of the tripod from beneath the beaker.
- Using a syringe, measure out 5ml of Trypsin into a test tube.
- Stand the test tube in the water bath and leave for several minutes until the enzyme has reached the desired temperature.
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Meanwhile, prepare the diluted casein suspension using 3cm3 of the substrate and 2cm3 of distilled water.
- Begin timing using stopwatch once the Trypsin has reached the desired temperature.
- Take the test tubes out at 5 minute intervals, and leave to cool in the test tube rack to room temperature.
- Mix the enzyme and substrate together. Observe the contents of the beaker by checking against the white card with the black cross on it.
- Record the time taken for the casein suspension to clear.
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Repeat this procedure 3 times in total for each time interval at temperatures 70, 80 and 90oC.
Risk Assessment
Trypsin; Handling and Storage
- Enzymes are biological active proteins and should be handled with care. Proteolytic enzymes such as Trypsin, even as a solution, may irritate the skin, eyes or mucous membranes. If prolonged skin contact occurs, it should be washed away with plenty of water. Safety should be considered when handling the enzyme.
- The Trypsin is made from a powdered preparation. The preparation is classified as harmful and you should avoid contact with the skin so protective clothing and gloves should be worn.
- When it is being prepared, adequate ventilation should be made available. Avoid inhalation of the powder as it may induce sensitisation.
- Clean up spillages immediately- do not allow them to dry and generate dust.
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The enzyme should be refrigerated between 0-4oC and protected from moisture.
Casein
Casein is in fact a low risk solution. However, it should be stored in a clearly labelled container in the refrigerator.
General Laboratory Safety
General safety should be applied when carrying out the investigation such as wearing appropriate protective clothing including gloves and goggles and tying long hair back. Increased care should be taken when using high-risk equipment such as Bunsen burners, water baths and glassware. Avoid the chance of spillages by keeping apparatus well away from the edges of workbenches. If any laboratory mishaps do arise, notify a member of staff immediately and warn those working around the area.
Consideration to the environment
No living organisms are being used in this investigation so there are no ethical issues. However, the enzyme must be disposed of in accordance with the Provisions of the Environmental Protection Act and should be followed down the sink with plenty of water. Unused enzyme must be re-used and stored appropriately. Resources should not be used in a wasteful manner.
Results
Raw Data
Time of clarification/s in Duration of Exposure of
Key: ________ those underlined are major anomalies and have been excluded from the averaging calculations, as these would obscure the results considerably.
Summary Tables
- Average times of casein clarification
Time of Clarification/s in Duration of Exposure of
- Average Rates of Reaction
Time of Clarification/s in Duration of Exposure of
Main Trends and Patterns
The results show that the increase in duration of exposure to high temperatures increased the rate of reaction. However, as the temperature increased, the rate of casein clarification and the rate of reaction were lower in general in comparison to the lower temperature in the experimental range.
This trend can be identified if you compare the time of clarification between the minimum duration of exposure value (5 min), against the maximum (25 min), at the experimental temperatures:
Time of Clarification/s in Duration of Exposure of
This comparison indicates that:
- The time of clarification increased with a greater exposure duration
- Time of clarification, as the temperature increased was lower in comparison to the lowest temperature.
Graph 1- A graph to show how the duration of exposure to high temperatures affects the time of clarification of casein
The graph presents the general trend associated with the affect of temperature on enzyme activity. The general trend is that as the temperature increases, the casein suspension clears at a faster rate. However, the average time of clarification of the casein suspension is lower as the temperatures increases in comparison to the lower temperatures.
Graph 2- A graph to show how the duration of exposure to high temperatures affects the rate of reaction of Trypsin on casein
I was able to calculate the rate of reaction using the following formulae:
Rate= 1 ____
Time taken (sec)
The graph shows that the general trend for all 3 temperatures is that duration of exposure increases the rate of reaction. However, the rate of reaction decreases as the temperature is increased.
This trend can be identified if you compare the rate of reaction between the minimum duration of exposure value (5min) against the maximum value (25 min):
Rate of Reaction at Duration of Exposure of
This comparison indicates that the rate of reaction increases with an increased in duration of exposure, although it is at a slower rate at the higher temperature. As a summary, although the time of clarification and the rate of reaction increases with an increase in duration of exposure, these two factors are lower in general as the temperature increases, in comparison to the lowest temperature in the range.
This trend can be developed through the theory of denaturation; as temperature increases, molecules and ions are gaining more kinetic energy, so there is an increase in collision frequency and the rate of reaction increases as a result. However, at temperatures, or above the optimum, (which we know is about 45oC), the delicate tertiary structure of the active site on the Trypsin molecule is destroyed. This is because there is an increase in kinetic energy and so the vigorous vibrations causes the bonds to break and the substrate molecule can no longer fit into the active site. Consequently, the high temperatures did have an effect on Trypsin denaturation.
It is also true to say that the denaturation of exposure time to high temperatures had an effect on Trypsin denaturation.
The results show a subtle change in that the gradients of the graphical lines on both graphs are gentle. This can be accounted for due to the fact that the Trypsin was allowed to cool down to room temperature after being heated. It also indicates that the Trypsin had a low turnover rate, which can be expected because the casein suspension was diluted and so the chance of collision is decreased as water molecules may get in the way. The Lock and Key Theory can explain this. In this theory it is assumed that the substrate will fit exactly into the active site. This means that the size, shape and specificity of the substrate correspond to the active site. In addition, it has also been found that the heat denaturation of enzymes is primarily due to the protein interactions with the aqueous environment. They are generally more stable in concentrated rather than dilute solutions.
To ensure that fair testing was maintained, the casein suspension was also kept at room temperature. Although the enzyme was reacted with the substrate at room temperature, the results obtained show that both the temperature and duration of exposure had an effect on Trypsin denaturation. However, the exposure to high temperatures seemed the most important factor in causing denaturation, as we know that the optimum temperature of most enzymes including Trypsin is 45oC.
Anomalous Results
When processing the results, some raw data had to be excluded from the averaging calculation, otherwise this may have obscured the results to a great extent. This can be seen at the duration of exposure of 10 minutes at 80oC- the highest value is 204.6s and the lowest is 133.8s. This is a large gap and if included in the anomaly, the value would have considerably flawed. The use of error bars is useful as it indicates how accurate a measurement is. As the error bar in this case span over a large area, it was considered as inaccurate.
An anomaly occurred at 70oC; where the duration of exposure of 20 minutes gave a time of clarification value of 79.2s. The lack of repeats for the 70oC results due to the limited time available makes this range unreliable. Another anomalous result occurred at 90oC; where the duration of exposure was 20 min resulting in a time of clarification value of 123.3s. Both anomalies do in fact fit in with the general trends, however were highlighted, as they would obscure the graphical lines. Therefore it can be assumed that they may have arisen due to the methods in which the investigation was pursed.
Evaluation
Through this investigation I was able to obtain a series of interesting conclusions, they cannot be 100% accurate due to the laboratory conditions and the allocation of time available to me. However, I am aware that this common as the tentative nature of all individual investigations. Within the school laboratory, it was impossible to maintain a completely sterile environment and therefore some degree of contamination could occur. In addition the method used could have accounted for the variability in my results. There are therefore improvements that could be made to the investigation to aid more accurate and consistent results.
Inaccuracies of the investigation
- Judging the end-point
- Temperature control
- Concentration of solutions
- Timing
- Enzyme preparation
- Judging the end-point
The end point concerned with this investigation was the time taken for casein suspension to clear- the end point indicates the completion of the reaction. This proved to be a limitation as it was based on eye judgement. This was not reliable as eye judgement and interpretation may vary respectively to each test and is therefore not constant.
Improvement
A colorimeter could be used to measure the achromatic point. This detects how much light has passes through a sample and so the clearer the solution, the higher the light transmission. Light transmission is measured as a percentage. This is an accurate method because it is able to measure to the nearest 2%, or even more accurately to the nearest 0.25%.
- Temperature control
-Thermometer
A spirit thermometer, which is the most inaccurate type, was used in this investigation. It is not sensitive enough to give an exact reading to measure the temperature of Trypsin, as it is only accurate to 0.5oC. A human error, which could have affected the results, was reading the temperature scale incorrectly. This could have been avoided by making sure that my eye level corresponded with the thermometer measurement to eliminate any chance of incorrect readings.
Improvement
To overcome the problem of human error, a digital probe thermometer could have been used. This is a more accurate alternative as it rounds off readings to two decimal places. In addition to this, it displays the readings so there will be no inaccuracies when reading the scale.
-Incubation methods
As high temperatures were needed in this investigation, water baths couldn’t have been used, as this isn’t energy efficient due to the high rate of evaporation that would have occurred. In addition, 30 oC, 40 oC and 60 oC were common temperatures used by pupils doing other investigation. Therefore, I had to set up a manual water bath heated by a Bunsen Burner. It was difficult to maintain a constant temperature due to its continual increase. Furthermore, as the test tubes were sitting in the beaker, heat from the Bunsen Burner would have been directly conducted through the glass, and so the thermometer may have measured the temperature of the bottom of the test tube and not the actual enzyme.
Improvement
The test tubes could be submersed into the manual water bath using stands.
- Concentration of solutions
-An unequal density gradient of the enzyme or substrate caused by setting may have affected the rate at which the Trypsin and casein reacts.
-Because of time restrictions, I often had to repeat the same reactions over a range of sessions, and so a new batch of diluted casein and the Trypsin was made up.
Consequently, the concentration of both solutions may not have been constant for each session.
Improvement
-Stirring before, during and after the exposure could have prevented the first problem to the specified temperatures. This is possible by creating a mini vortex using a stirring rod to achieve a homogeneous or equimolar solution, ensuring the solution does not set.
-To solve the second problem, I should have created a single batch of diluted casein from which work throughout the duration of the investigation and also kept aside a sample of Trypsin to ensure concentrations of both solution remain constant.
- Timing
Due to the tentative nature of this experiment, and in fact all individual investigation in general, time proved to be a limiting factor. As a result, the tight time frame in which to carry out the investigation contributed to the lack of repeats for the 70oC test, and was therefore made unreliable. To ensure that I obtained accurate averages in the restricted time frame, I often done three reactions at one time. A problem with this was that I forgot to look at the stop clocks or I did not stop them on time, so the time of exposure at a particular temperature ran over.
Improvement
To solve this problem, I could have used stop clocks with alarms on them to alert me when specified time intervals were over, or near to completion.
- Enzyme preparation
Many enzyme preparations follow a series type deactivation scheme and in some cases, may be due to structural micro heterogeneity, where the enzyme preparation consists of a mixture of a large number of closely related structural forms. This may have occurred during preparation or storage due to a minor reaction, such as deamidation of one or more residues, limited proteolysis or disulphide interchange. Alternatively, it may also have been due to quaternary structure of the enzyme molecule.
Improvement
In order to minimise the loss of activity on storage, even moderate temperatures should be avoided. Most enzymes are stable for months if refrigerated (0-4oC). Cooling below 0oC in the presence of additives, which prevent freezing, can increase storage stability. Freezing solutions causes denaturation due to the stress and pH variation in ice-crystal formation, and so must be avoided.
Extension to the Investigation
Alternative Method
The significant aspect of an extension to this investigation is the method that was used. Due to the tentative nature of individual experiments and classroom experiments as a whole, it proved to be difficult to control external factor such as the regulation of temperature and duration of exposure, degree of setting etc. however such factors can be controlled more accurately with the use of a thermostatic magnetic stirrer. With the use of such equipment, high temperatures can be used and maintained by pre-setting the dial. The concentration distribution whilst heating and when reacting when cooled down to room temperature is able to remain constant due to the current created by the magnetic follower, so more emphasis is then able to be directed to judging the achromatic point. Overall this method is also less time consuming and so would be more suitable when working within a tight time frame.
Further Work
It was found that both exposures to high temperatures, and the duration of exposure had effect on Trypsin denaturation. However, the degree of each was not accurately resolved. Repeating this investigation at a lower range of temperatures, such as between 10 and 50oC can closely follow the degree on duration of exposure on Trypsin denaturation alone. Furthermore, it has been discovered that the heat denaturation of enzymes is primarily due to the protein interactions with the aqueous environment. They are generally more stable in concentrated rather than dilute solutions, so concentration of the Trypsin preparation can be increased to eliminate this effect.
Trypsin is sensitive to pH, and so the effect of pH on its activity can be investigated. Excessive acidity or alkalinity renders the enzyme inactive. However, certain digestive enzymes prefer a distinctly acidic or alkaline environment. Hydrocarbon ions make pancreatic juice slightly alkaline (pH 7.1 to 8.2), allowing it to neutralise stomach acids and to create optimum conditions for the digestive enzymes. The pH of the medium can have direct affect on the bonding responsible for the secondary and tertiary structure of the enzyme. A suitable range of buffer solutions can be adopted for this extension to investigate its effect on the activity of Trypsin.