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Investigate what effects the duration of exposure to a high temperature has on enzyme denaturation.

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Biology Individual Investigation- Enzymes Aim: To investigate what effects the duration of exposure to a high temperature has on enzyme denaturation. Introduction: Living cells carry out many biochemical reactions. These reactions take place rapidly due to the presence of enzymes. Enzymes are biological catalysts that speed up the rate of reaction without being used or destroyed. Enzymes also have the ability to control reactions. The ability of an enzyme to function depends on the specific shape of the protein molecule. The intricate shape created by the polypeptide folding is a key factor in the theories of enzyme action, in which temperature has a great effect. Trypsin Trypsin is the enzyme that will be used in this investigation. Trypsin is an endopeptidase enzyme, or pancreatic serine protease with substrate specificity based upon positively charged lysine and arginine side chains. Its optimum pH is 8.0. Trypsin is a constituent of pancreatic fluid, splitting complex proteins into simpler components that can be absorbed and used in reconstructing body proteins. Although Trypsin can begin the digestion of a wide variety of foods rich in protein; for example eggs, pork, chicken and Soya, it is specific. The hydrolysis of Trypsin is limited to bonds formed by either lysine or arginine residue. Most proteins have these two amino acids next to each other at some points in their polypeptide chain, and so Trypsin is able to partially digest it. Substrate-Casein Organophosphorus compounds such as diisopropylflurophosphate and natural inhibitors from the pancreas inhibit trypsin. Soybean, lima bean and egg whites are also sources of natural inhibitors including Casein, a protein precipitated when milk is mildly acidified. When hydrolysed, the suspension begins cloudy, but becomes clearer as the products dissolve. This reaction is catalysed by Trypsin, producing amino acids as the reaction product. The effect of the duration of exposure to a high temperature on Trypsin denaturation can be noted by the changes in clarity of the casein suspension. ...read more.


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. * 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 Temp/oC at which Trypsin was incubated 5 min Average / s 10 min Average /s 15 min Average /s 20 min Average /s 25 min Average /s 70 123.11 123.11 87.61 87.61 79.21 79.21 75.62 75.62 68.41 68.41 80 142.12 ...read more.


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. ...read more.

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