Investigating how pH effects the enzyme trypsin acting on coloured gelatin free essay sample

The results from the experiment determining the effects of pH on enzyme activity show that as the independent variable, pH, increases the dependent variable, percentage transmission, decreases. This is shown in the results as at the lowest pH, pH 4. 0 the average percentage transmission is at its highest at 97%. At the highest pH, pH 8. 0 the average transmission is 78. 5%. This is also supported from the graph as it produces a negative gradient showing that as the percentage transmission will decrease with an increasing pH. This happens because the enzyme trypsin acts on the gelatine. Therefore as the pH increases towards the optimum pH more jelly will be broken down by the enzyme, allowing less light to pass through the solution which would thus decrease the percentage transmission. Trend: The trend in the results and from the graph show that the more the pH increases the lower the percentage transmission will be. However from looking at the results an optimum pH is unidentifiable. We will write a custom essay sample on Investigating how pH effects the enzyme trypsin acting on coloured gelatin or any similar topic specifically for you Do Not WasteYour Time HIRE WRITER Only 13.90 / page This is because the range of pHs used in the experiment is limited from 4. 0 to 8.0; the percentage transmission may have continued to decrease as the pH became more alkaline. On the other hand from the pHs used we can deduce that the optimum is 8. 0 as it gives the lowest percentage transmission which shows that the enzyme is more effective in this condition and breaks down the jelly more effectively than when it is in the solutions of lower pH values. This agrees with the hypothesis as it stated; â€Å"Most digestion and most colour release from the jelly would be expected at a pH of 8 and in solutions above or below this pH less colour should be released. † Biological evidence to support results: The relationship shown between the percentage transmission and pH occurs as the enzyme trypsin works best in more alkaline conditions. This is because it is found in the body in the duodenum where the pH is alkaline, which supports the results as in this experiment the pH was 8. Therefore as the pH increases more gelatine will be broken down because towards the optimum the active site of trypsin best facilitates the formation of the enzyme-substrate complexes as the active site will be the optimal shape for attachment. Less light can pass through the solution due to the gelatine being hydrolysed, which is the breaking of the peptide bonds between the NH2 and the COOH resulting in these forming groups on the amino acids. If more gelatine is hydrolysed there were be a greater amount of colour released from the jelly. At a lower pH such as pH 4 or 5, the more acidic conditions reduce the enzyme activity. This is because the structure of the protein and therefore the active site of the enzyme are altered by changes in pH. In particular ionic bonds, hydrogen bonds and disulphide in the tertiary structure may be disrupted, this can cause and unravelling of the tertiary globular structure, the enzyme is said to be denatured. So at non-optimal pH the substrate attaches less readily to the enzyme as the active site is no longer complementary to the substrate. It is only the optimal when the active site will best facilitate the enzyme-substrate complex formation. It is the unique R-groups of the amino acids that form a particular structure and the active site. There is therefore a specific active site formed by the R-group that best facilitates the bonding of the substrate to the trypsin. Evaluation: For measuring the percentage transmission results a colourimeter was used. This is because eye judgement is insufficient and using the colourimeter provides quantitative values. Due to the red pigmentation of the gelatine and trypsin solution a blue filter must be used in the colourimeter. A blue filter transmits blue light. Blue is also at the opposite end of the spectrum to the red, the blue light is then absorbed by the red solution and a reading can be taken. The pipette and pi-pumps were used to transfer the buffer solutions, distilled water and the trypsin into the boiling tubes. These were used as the pipette is the most accurate way of ensuring the exact amount of solution is used. When transferring the solutions into the boiling tubes you must touch the surface of the solution with the bottom of the pipette, this means that all the solution is added to the tube and therefore will increase the reliability. To improve the results from the experiment buffer solutions that were not whole pHs could have been used e. g. pH 4. 5, 5. 5 etc. This would have provided more reliable results as a wider range of results would have been produced. Using pHs with decimals would also help to more accurately determine the optimum pH as the optimum may have been above or below the pH stated in the hypothesis; 8. In this experiment however the optimum is taken at 8 because the graph does not rise again. To ensure the experiment was kept as a fair test a number of variables were controlled. The temperature of the solutions was kept constant by placing the boiling tubes into a test tube rack and setting it into a water bath with a fixed temperature of 25oC. The temperature needed to be kept low and fixed as a high temperature would denature the enzymes, they would therefore be unable to break down the gelatine and no results would be produced from the experiment. Keeping a constant temperature also meant that the solutions reacted at the same rate. The time in the water bath was also controlled to ensure that the enzymes were left to react for the same amount of time, making the experiment fair. If the enzymes were not exposed to the temperature for long enough then they would not have reacted well enough to produce valid results. The enzyme concentration used was a 2% concentration of Trypsin. If a higher concentration had been used in some of the boiling tubes the rate of the reaction would have increased. This is because there would have been more available active sites for the substrate to bind to; forming enzyme-substrate complexes at a faster rate and therefore more of the jelly would have been broken down during the time. Whereas if a lower concentration was used the active sites would be saturated and the rate would decrease. When cutting the jelly cubes the size needs to be uniform for the cubes. If the cubes are too small there isn’t a sufficient amount to be broken down by the Trypsin, causing the results to be unreliable. If they are too large then they may not completely dissolve in the solution meaning that they will block the light passing through the solution in the colourimeter. Validity of Results: Due to the wide range of results they are not reliable as there is no narrow range and there are many areas of the experiment that could have caused a decrease in the reliability of the results. The jelly cubes were not cut to the exact same size. This would have caused a decrease in the rate if they were too small. It could have been improved by measuring the lengths of the sides of the cubes and weighing them to find out their mass. A series of precautions must also be taken using the colourimeter. The cuvettes must be clean with no finger prints. If they are dirty it will stop light passing through the cuvette to the colourimeter making the results unreliable. The same cuvette must also be used throughout and it needs to be orientated in the same way. The cuvette must be filled with sufficient solution to ensure the light is intercepted. If there is not enough solution the light will pass straight through producing a high unreliable reading. Assessment of Pooled Results: pH Lowest Percentage Transmission Highest Percentage Transmission Range 4. 0 77 99 22 5. 0 70 93 23 6. 0 64 94 30 7. 0 60 89 29 8. 0 58 85 27 The pooled results provide a wide range of results that makes the final results unreliable. There is a large range constant throughout the results. There are also some large differences between repeated results. It should have been expected that the pooled results would have been reliable as there are essentially 9 repeats. The main reason the data is unreliable is because the jelly cubes were cut too small and across the different groups there would be no uniform size of cubes. The small cube size would affect the results because of the small surface area. The Trypsin would have had enough time to break down the gelatine. There also should have been a greater spread of pH between 4. 0 and 8. 0. If the experiment was to be repeated larger jelly cube sizes would need to be used to improve the reliability.