Enzymes: Lab Report

Introductory Biology 1 Biology 1003 Fall Term 2011 Lab Number: 3 Title: Cell Energetics: Enzyme Role in Biological Reactions Name: Brandon Moore Student Number: 100819124 Lab day and time: Wednesday pm Date: Wednesday November 23, 2011 Introduction Enzymes are a key aspect in our everyday life and are a key to sustaining life. They are biological catalysts that help speed up the rate of reactions. They do this by lowering the activation energy of chemical reactions (Biology Department, 2011). In chemical reactions bonds must be broken and new bonds must be formed. In order for this to occur the bonds must be made less stable. For bonds to become less stable a small input of energy is required and this is called the activation energy. In simpler terms, in order for a reaction to begin and proceed spontaneously a small input energy is required to give the reaction a push and get it started (Cooper, 2000). As said before catalysts are chemical agents used to speed up the rates of reactions. The biological catalyst is a group of proteins called enzymes. Enzymes work by lowering the activation energy and making it easier for the eactants to obtain the necessary energy to break the kinetic barrier. Even though enzymes speed up the rate of reaction, they do not change the free energy of the reactants and the products (Russel et al. , 2010). Enzymes work by combining with reacting molecules at the active site. Each enzyme is specific to only one kind of molecule and can only bind to its specific molecule. The active site is a groove in the enzyme wher e the molecule will bind to; this is formed by the enzyme folding into a specific shape. When the enzyme is done and the molecules are then in the transitional state, which means the bonds are unstable and ready to be broken, the enzyme remains unchanged and can continue to bind to other molecules (Russel et al. , 2010). Enzymes induce the transition state by three major mechanisms. The first is by bringing the reacting molecules together. The reactants bind in the active site of the enzyme in the right orientation for catalysis to occur. The second mechanism works by the enzyme exposing the reactant molecule to altered charge environments. The third mechanism is by changing the shape of a substrate molecule (Russel et al. , 2010). The conditions being studied on how they affect enzyme activity are: concentration, ph, and temperature. As the concentration of enzymes increases the rate at which products are formed also increases. It is also true as the concentration of the substrate increases the rate of the reaction will also increase until the enzymes reach their maximum rate at which they can combine with the substrates. Each enzyme has a best possible pH where it works at its best. Anything that changes on either side of the optimum pH will decrease the rate of the reaction. Finally as temperature raises so does the rate of the reaction but only to a certain point. As the temperature raises the frequency and strength of collisions will increase, however if the temperature rises too high the hydrogen bonds of the enzyme break and it unfolds making it unable to accept any molecules due to its active site being destroyed. To observe the effects of these three conditions on enzyme activity spectrophotometry is used. A spectrophotometer works by measuring the amount of light a compound in solution absorbs. As the concentration of the solution increases more light is absorbed (Biology Department, 2011). The purpose of this experiment is to test and observe the effects of concentration, pH, and temperature on enzyme activity. Methods In part I of the lab obtain six small glass tubes in a test tube rack. After the six small tubes are obtained, add fifteen drops of distilled water to tube 1, ten drops to tube 2 and 3, five drops to tube 4, and no drops to tubes 5 and 6. Once distilled water is added five drops of the substrate solution were then added to tube 2, 4 and 6. There were no drops of substrate solution added to tubes 1 and 3, and ten drops were added to tube 6. After the substrate solution was added, five drops of the enzyme were quickly placed in tubes 3, 4 and 5. There were no drops of enzyme added in tubes 1 and 2 and in tube 6 ten drops were added. Once the enzyme solution has been added the tubes were then left to incubate for ten minutes and after five drops of DNSA solution were added to tubes 1 to 6. The tubes were then placed in a hot block at 80-90oC for five minutes. They were then taken out after the five minute period and using a 5 ml pipette, 5 ml of distilled water were added to the 6 tubes and mixed by inversion. Once everything was complete the 6 tubes were then taken to the Milton Roy Company Spectronic 21 and the absorbance of each tube was tested. In part II of the lab six small glass tubes were obtained in a test tube rack. Ten drops of distilled water were then added to test tube 1, five drops to tubes 2-4, and no drops in tubes 5 and 6. Five drops of 0. 1M HCl were added to test tube 5 and five drops of 0. 1M NaOH to test tube 6. Five drops of enzyme were then added to all tubes except tube 1. Tube 3 was then placed in the ice bucket and tube 4 was placed in the hot bucket at 80-900C for five minutes, the remaining tubes were left in the test tube rack. After the five minutes five drops of 1% starch was added to every tube and left to sit for ten minutes. After ten minutes five drops of DNSA were then added to all the tubes. All the tubes were then taken and placed in the hot bucket at 80-900C and left to incubate for five minutes. After the five minutes, take a 5 ml pipette and add 3 ml of distilled water to each tube and mix with inversion. Once everything is complete the tubes were then taken to the Milton Roy Company Spectronic 21 and the absorbance of each tube was tested. Results In part I tubes 1-3 had a very low absorbance. In tube 4 when the enzyme and substrate were present the absorbance increased substantially from below 0. 1 to a mean of 0. 53. When two times the amount of substrate was added in tube 5 the absorbance increased again from a mean of 0. 53 to 0. 57. Finally when two times the amount of enzymes was added the absorbance increased a final time from 0. 57 to 0. 63. Table 1. The effects of different concentrations on the absorbance of solutions Lab Group |Tube 1 Abs. |Tube 2 Abs. |Tube 3 Abs. |Tube 4 Abs. |Tube 5 Abs. |Tube 6 Abs. | |Our Group |0 |0. 05 |0. 09 |0. 55 |0. 68 |0. 66 | |Group 2 |0 |0 |0 |0. 61 |0. 725 |0. 75 | |Group 3 |0. 01 |0. 02 |0. 01 |0. 42 |0. 3 |0. 49 | |Mean |0. 0033 |0. 023 |0. 33 |0. 53 |0. 57 |0. 63 | |SD |0. 0058 |0. 025 |0. 049 |0. 097 |0. 23 |0. 13 | |SE |0. 0033 |0. 015 |0. 029 |0. 056 |0. 14 |0. 076 | Tube 1 was the control and recorded a low absorbance of approximately 0. 01. Tube 2 contained the enzyme and substrate and the absorbance rose to a mean of 0. 54. When tube three was heated and tube 4 was cooled the absorbance ecreased to 0. 32 and 0. 38. Finally solution of 0. 1M HCl was added to tube 5 and the absorbance decreased to 0. 0025, and solution of 0. 1M NaOH was added to tube 6 and the absorbance decreased to 0. 13. Table 2. The effects of pH and temperature on the absorbance of different solutions |Lab Group |Tube 1 Abs. |Tube 2 Abs. |Tube 3 Abs. |Tube 4 Abs. |Tube 5 Abs. |Tube 6 Abs. | |Our Group |0 |0. 63 |0. 39 |0 |0 |0. 4 | |Group 2 |0 |0. 15 |0. 9 |0 |0 |0. 01 | |Group 3 |0. 05 |0. 85 |0. 49 |0. 11 |0. 01 |0. 08 | |Group 4 |0 |0. 54 |0. 31 |0. 04 |0 |0. 03 | |Mean |0. 013 |0. 54 |0. 32 |0. 038 |0. 0025 |0. 13 | |SD |0. 025 |0. 29 |0. 17 |0. 52 |0. 005 |0. 18 | |SE |0. 013 |0. 15 |0. 085 |0. 026 |0. 0025 |0. 091 | Discussion Enzymes are biological catalysts that reduce the activation energy in order to increase the rate of the reaction. Increases in concentration increase the rate of the reaction, change in pH from the optimum will decrease the rate of a reaction, and increasing temperature will also increase the rate of reaction until a certain point is reached (Worthington Biochemical Corporation. 1972). Part I of the lab focused on the effects of concentration on pH. When we look at table I we can see that tubes 1-3 had very low absorbances. Tube 1 was the control that contained only water and no reaction occurred. In tube 2 the enzyme was not present which meant that the reaction occurred spontaneously without any help, thus a low absorbance. Tube 3 contained the enzyme but lacked the substrate, which meant nothing was bonding to the active sites and reaction could not occur. In tube 4 both substrate and enzyme were present and the absorbance rose greatly from approximately 0 to a mean of around 0. 3. This perfectly demonstrates that with the addition of an enzyme the product concentration increases and so does the rate of reaction. To tube 5, two times the amount of substrate was added and absorbance increased again to a mean of 0. 57. This shows that more substrate was present and readily available to bind to the active sites. Last was tube 6 which contained two times the amount of enzyme and again the absorbance rose to approximately 0. 63. The increase of enzymes allowed for more active sites to be readily available to bind to the molecules (Worthington Biochemical Corporation. 1972). When viewing the data obtained and comparing it to what is known about concentration effects on enzyme activity it can be accurately concluded that the data obtained is fairly accurate. As the enzyme concentration is kept the same and the substrate concentration increases the rate of reaction will also increase. This makes sense since now there are more molecules of substrate available to bond to the active sites. Increasing concentration will only increase the rate of reaction until a certain point is met. This point occurs when too much substrate is added and all available enzymes are already working. When this occurs the concentration increase no longer has an effect on the reaction rate. This is also true with the increase in concentration of the enzyme. The more enzymes there are the more active sites available to bond to the molecules. The increase in enzyme concentration will also increase the rate of reaction. This concludes effectively that the data obtained effectively demonstrates the effects of concentration on the rates of reactions (Worthington Biochemical Corporation. 1972). Part II of the lab focused on the effects of temperature and pH on enzyme activity. When viewing table II it can be seen that tube 1 had a very low absorbance, due to it being the control and not containing any substrate or enzyme. Tube 2 contained the substrate and enzyme and thus the absorbance increased greatly to a mean of 0. 54. When looking at the changes of pH in tubes 5 and 6 the absorbance decreased for both to 0. 003 and 0. 1. The optimum pH is around 7 and with this the reaction rate is at its best. As stated before any change in pH away from the optimum will decrease the rate of reaction. HCl has a lower pH than 7 and is below optimum, which means that it will have more unstable charges and the absorbance will lower, which is what was seen in tube 5. The same happens for NaOH, which is on the other side of the pH spectrum and above the optimum pH of 7 as seen in tube 6. From this it can be concluded that any change in pH away from the optimum will cause an unbalance in charges and cause the reaction rate to decrease (Worthington Biochemical Corporation. 1972). The second part of part II involves the effects of temperature. When looking at tube 3 that was placed in the ice bucket the reaction rate decreased from tube 2 with mean absorbance of 0. 54 to a mean of 0. 32. A decrease in temperature will slow down the activity of the substrate and enzymes and will reduce the speed and amount of collisions occurring. With less collisions occurring the reaction rate will then decrease. Tube 4 was placed in heat and the absorbance dropped as well to a mean of 0. 38. Stated before it was said that an increase in temperature would cause the speed and number of collisions to increase. This would then increase the rate of the reaction. However, an increase in heat will only increase the rate of reaction until a certain temperature is reached. This temperature is approximately between 40-50OC. Tube 4 was placed in temperatures ranging from 80-90OC, which is much higher than the max of 40-50. When this max is surpassed the hydrogen bonds will begin to break and the enzymes will unfold. When the enzyme unfolds the active site will then be destroyed and become deformed and no longer usable. When this happens the enzymes stop functioning and the reaction rate will decrease, which is what was seen (Worthington Biochemical Corporation. 972). The living cell is a site for activity known as metabolism. This can include the build-up or repair of tissues, turning food into energy, getting rid of waste products, and all the activities of life. Many of these processes do not occur spontaneously and this is why enzymes are needed. Without enzymes life itself would not be possible (Cooper. 2000). It can be concluded that concentr ation, pH, and temperature have great effects on enzyme activity. The increase in concentration of substrates increases the reaction rate until the point where all enzymes are being used. The increases in enzyme concentration will increase the rate of reaction. Any change in pH away from the optimum will cause an unbalance in charges and will lower the reaction rate (Worthington Biochemical Corporation. 1972). Finally the increase in temperature will increase the reaction rate until around 40-50OC when hydrogen bonds begin to break (Russel et al,. 2010). By understanding more about enzyme catalysts advances in medicine and life sciences are able to occur and help us understand more about life itself. References: Russell, P. J. , S. L. Wolfe, P. E. Hertz, C. Starr, M. B. Fenton, H. Addy, D. Maxwell, T. Haffie, and K. Davey. 2010. Biology: Exploring the Diversity of life, first Canadian edition. Nelson Education Ltd. , Toronto. Biology Department. 2011. Introductory Biology: BIOL 1003 Lab Manual. Carleton University Press, Ottawa. Worthington Biochemical Corporation. 1972. Introduction to Enzymes. http://www. worthington-biochem. com/introbiochem/effectspH. html. November 22, 2011. Geoffrey M Cooper. 2000. The Cell: A Molecular Approach, Second Edition. Sinauer Associates Inc, Boston University.