Effect Of Temperature On The Digestive Enzyme Pepsin Biology Essay

Effect Of Temperature On The digestive Enzyme Pepsin Biology EssayIn this study an taste was carried surface to coiffe if varied temperatures actuate the deem at which enzymes function. Enzymes be biological catalysts catalysts are substances that increase the assess of chemical reactions with surface being used up (BBC, 2010), without these catalysts it would maintain an extremely long time for these reactions to take place. The enzyme used in this bad-tempered experiment was pepsin pepsin is a zymogen of pepsinogen. Pepsinogen is activated by hydrochloric acid, which is released from parietal cells in the stomach lining. The hormone gastrin and the vagus nerve trigger the release of both pepsinogen and hydrochloric acid from the stomach lining when food is ingested. Hydrochloric acid creates an acidic milieu, which allows pepsinogen to exsert and cleave itself in an autocatalytic fashion, generating pepsin. (Life Science Net body of work, 2010)A lot open fire be learn t about enzymes by studying the value of enzyme catalyzed reactions, these rates of reaction net be studied in various ways. In this experiment, apply a range of different temperatures, the enzyme pepsin will be mixed with screwball albumen. This is high in protein and bound to the dye Coomassie blue to gain a readable absorbance reading using a spectrophotometer and in topic charm how much protein has been digested by the pepsin.Egg albumen was used as the protein out practise in this study as although it is composed mainly of around 80% water it has about 15% of its total mass made up from approximately 40 different types of proteins, mainly Ovalbumin (54%) (Edin Formatics, 1999).A spectrophotometer is a device used for measuring scintillation intensity and will be used to determine the amount of protein in separately mixture, it works by measuring the timid intensity as a function of the comment or more than specifically the wavelength of light (Global Water Instrume ntation Inc, 2007) ( auxiliary 2). thereof the lower the reading means less light has been absorbed by the solution being raveled indicating in this case that more protein (egg albumen) has been digested by the enzyme (pepsin) and the lower the reading the faster the enzyme reaction rate.If enzyme reactions are affected by temperature, and so changes in temperature may bring about different absorbances of light readings related to how much protein has been dispirited down by the enzyme. As pepsin is erect in the stomach it would seem only logical to assume the best temperature for this particular enzyme would be around 37c, humane body temperature. ruleA cuvette was filled with 0.5ml of di composeed water and placed in a spectrophotometer, then the machine was calibrated by pressing the zero button with the spectrophotometer set to a wavelength of 595nm. aft(prenominal) calibration 0.5ml of egg albumen (2mg.cm-) was added to a test metro using a glass pipette and incubated in a pre heated water bath at a temperature of 10c (then 20c, 30c, 40c, 50c, 60c, 70c) for 5 minutes.0.5ml of pepsin (0.1%) was then added to the test tube and incubated at the same temperature for a advertize ten minutes.After this incubation period 2.0ml of Coomassie blue reagent was added to the test tube and immediately mixed by capping the test tube with parafilm and inverting several times.After the test tubes contents had been mixed they were carefully poured into a cuvette and placed into the spectrophotometer with the light absorbance levels being recorded at a wavelength of 595nm.The experiment was carried out common chord times at each temperature to achieve reliable data.ResultsThe results documented in Table 1 are the light absorbance reading averages of trinity dampen experiments carried out at each temperature. A full set of results elicit be seen in Appendix 1. Figure 1 exposes the averaged results plotted on a scatter graph.Table 1Temperature (Degrees Celsius )Average light absorbance at 595nm102.501202.550302.516402.403502.543602.740702.806Fig 1 Change absorbanceDiscussionThe results in Table 1 as well up as the bell curve graph (Fig 1) show that the best temperature as predicted in the hypothesis seems to be 40c, constraining to human body temperature. These results also show that temperature has a definite instal on the rate the enzyme reacts to breakdown the protein in the egg albumen.Table 1 shows that at 10c, 20c and 30c the light absorbed by the solution is more than at 40c, this is because the pepsin has not broken down as much of the egg albumen at lower temperatures as it has at 40c so the spectrophotometer is woof up more undigested protein particles in these readouts. The same applies to the temperatures above 40c and indicates 40c is the best temperature range for pepsin to be brisk.Enzymes are made up of amino acids amino acids are the basic construct blocks of proteins consisting of a basic amino group, a carboxyl group, a heat content pinch and an organic side group attached to the carbon atom (Biology Online, 2010). When an enzyme is formed it is made by stringing together between 100 to 1,000 amino acids in a specific and unique order defining the three dimensional shape of the enzyme and its particular chemical reactivity (Brain, M. 2000).The lock and make supposition explains how an enzyme may work, it utilizes the concept of an active site on the enzyme. The theory is that a particular part of the enzymes surface has a strong affinity to the substrate (protein). The substrate is held in such a way that its changeover to the reaction products is more favorable. If you consider the enzyme is the lock and the substrate is the key out, the key is inserted in the lock and turns it to open the door letting the reaction proceed (Worthington biochemical Corporation, 2010) (Appendix 3). However, the induced fit theory expands on the rigid lock and key theory. This updated view of enzymol ogy proposes that the substrate causes a conformational change in the enzyme so the active site achieves the exact configuration for a reaction to occur, the overall effect being a tighter medical dressing between the enzyme and substrate (Allaby, M. 1999) (Appendix 4). The benefit of this tighter fecundation would be a faster reaction rate as more surface area of the enzyme would be in contact with the substrate.The energising concussion theory describes temperature affects on a system as the amount of energizing vital force it has, a lower temperature will provide less kinetic energy than a higher temperature. When molecules collide the kinetic energy can be converted into chemical potential energy, if the chemical potential is commodious enough the activation energy or energy required for an enzyme to work can be chafeed. The more chemical potential energy molecules scram when they collide, a greater number of molecules per unit time will reach the activation energy neede d to bind the enzymes active site to the protein resulting in a quicker rate of reaction. If the temperature gets too high some of the weak bonds that determine the shape of a protein and its active site could be broken resulting in the enzyme becoming denatured and decreasing the rate of reaction sometimes rendering the enzyme inactive (Brooklyn College, 2010). Figure 1 shows that after 50c the enzyme reaction rate slows down considerably, the enzyme is denaturising at a faster rate than it is below 30c. This change in enzyme reaction rate may be due to the fact that pepsins are stored at low temperatures to interrupt the enzyme destroying itself, therefore pepsin is less active at lower temperatures until it reaches its activation energy around 30c and boththing beyond around 50c 55c will rapidly denature the pepsin so the molecules in the active site can no longer bind to the protein and pass water a reaction, rendering the enzyme inactive permanently.Once the pepsin has dig ested the egg albumen it would still be difficult to analyse the amount of protein left at each temperature, this is why Coomassie was added before taking a reading. In an acidic environment the protein will bind to Coomassie causation a spectral shift from a reddish/brown colour with a low absorbance maximum of 465nm to a light blue colour with a higher maximum absorbance of 610nm with the leaving of the two colours greatest at 595nm, an optimal wavelength (Thermo Fisher Scientific, 2010). The binding of the Coomassie takes place when the red form donates its free electron to the ionisable groups on the protein causing a disruption of the proteins normal state and revealing its hydrophobic pockets. These pockets, via cutting edge der Waals forces (attractive and repulsive forces between molecules) bind to the non polar region of the dye, putting the autocratic amine groups close to the negative charge of the dye, creating a strong bond. binding of the protein stabilizes the bl ue form of Coomassie dye, thus the amount of complex present in solution is a measure for the protein concentration by use of an absorbance reading. (Bradford M, 1976, P248-254)Although this experiment has produced reliable accurate data that has proven the hypothesis right, many things could affect the results and readings obtained. When using such an accurate way of recording the data such as a spectrophotometer a number of things could affect the reliability of the results. Things alike minor differences in volumes of substances added to the test tubes or inattentive timing methods could be damaging to the results obtained. Simple human error could possibly influence any results with any fingerprints or water on the cuvette affecting the absorbance readings.ConclusionIn conclusion, the study carried out was adequate for the data required and indicated that temperature definitely affects the rate at which an enzyme reacts. As long as the method is executed well this is a great ex periment to look at temperature and its effect on enzyme activity however as with any scientific study human input is a crucial factor and could affect the quality of results. other experiment may need to be carried out to determine what the optimum temperature is on a more specific scale, something closer to body temperature would booster to discover a more precise optimum temperature, 35c 40c for example. As well as finding out an exact optimum temperature a further study to find out the optimum pH of pepsin could be done to further enhance the enzymes rate of reaction, focused around the acidic pH in the human stomach.AppendicesAppendix 1Temperature (c)Absorbance at 595nm (Reading 1)Absorbance at 595nm (Reading 2)Absorbance at 595nm (Reading 3)102.4302.5502.520202.4802.5302.640302.5002.5102.540402.3602.4002.450502.5202.5602.550602.6602.7802.780702.8002.8202.800Appendix 2http//commons.wikimedia.org/wiki/ archiveSpetrophotometer-en.svgAppendix 3http//www.chemistry.wustl.edu/edud ev/LabTutorials/Carboxypeptidase/images/lockkey.jpgAppendix 4http//wpcontent.answers.com/wikipedia/commons/thumb/2/24/Induced_fit_diagram.svg/450px-Induced_fit_diagram.svg.png