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An Investigation into the Effect of Varying pH on Enzyme Activity

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Introduction

An Investigation into the Effect of Varying pH on Enzyme Activity Introduction Protease is an enzyme that reacts with protein to break it down into its constituents. Enzyme activity can be affected by the change of pH, temperature and the concentration of substrate or enzyme in the solution. The focal aim of this experiment is to see what pH our protease works at best to determine where it is made in the body. The casein used comes from marvel milk and is a protein. It is a protein that is present in milk to give it its white colour. When broken down into amino acids, the milk becomes clearer. When the protease reacts with the casein, it turns clearer from its original cloudy colour. The reaction therefore would be monitored using a colorimeter that measures the amount of light going through the solution and the more light that is let through, the more the amount of substrate has been broken down. However the colorimeter uses a filter to only let some wavelengths of light through or else it would let all the light through not showing distinctions between different solutions. Therefore a filter must be used and as the come in different colours letting only some wavelengths of light through so the most suitable was chosen for the experiment which was 490 nm. (A filter was not necessary for the experiment but one had to be used as the colorimeter would not work without one.) The buffer solutions are solutions that resist changes in their pH, even when small amounts of acid or base are added. They are said to be amphoteric. So if 8.8 pH buffer solution is added, it would mean that the total pH of the solution is 8.8 and they keep the pH constant but if the solution gets acidic to the pH of 8.9, it would remove some hydroxide ions to get the pH back to 8.8. ...read more.

Middle

increases in acidic conditions, it will determine the acid is made in the stomach and in the duodenum if it works best in alkaline conditions. Results In the experiment, three repeats were done for each different buffer solution. They were written down and the average time was found out. The rate of reaction was also found by dividing 1 by the average time taken and then multiplied it by 1000 to make it easier to plot on a graph. Analysis Looking at the graph very strong positive correlation can be seen showing that as the pH increased, the rate of reaction increased (The positive correlation is represented with a red oval). The graph also shows that the enzyme used has the optimum pH of 8.8 as the rate of reaction is the highest (58.8). An enzyme is a globular protein molecule which in a biochemical reaction as a catalyst. Enzymes can be intracellular (work within a cell) or extra cellular (work outside a cell). The enzyme used here was trypsin. This conclusion was reached using scientific knowledge and research. Enzymes are very sensitive to pH changes and they all have an optimum pH where they work best at. The enzyme used in this experiment worked best at pH 8.8 showing that its optimum pH was alkaline suggesting firstly that it was trypsin. Looking at the graph it can be also deduced that the trypsin did not work best at the acidic conditions. This suggests that if extreme pHs were to be used (lesser the pH of 5.9) the trypsin would not work. This is due to there being many free H+ ions (in too acidic solutions) and OH- ions (in too alkaline solutions) thus disrupting the ionic bonds, that maintain the tertiary structure and denaturing the enzyme and changing the shape of the active site therefore no reaction happening as the substrate will not fit as readily into the active site. ...read more.

Conclusion

Higher temperatures mean that more kinetic energy is given to the particles making them move around more and quicker making more successive collisions are possible thus increasing the rate of reaction. The colorimeter was very accurate and sensitive and was affected by light from the environment around so the experiment was done on the same day to ensure that the climate and conditions were the same. However, it is a possibility that the slight changes in light would have changed the amount of light transmitted through therefore increasing the chance of human error. Another aspect that could be considered is the use of a flea (magnetic stirrer) that would mix the solution more uniformly for each experiment ensuring more reliable results. In the experiment the testube was shaken for each repeat as evenly as possible but it was not as accurate as using a magnetic stirrer and therefore due to human error the shaking would have affected our results showing a change in the rate of reaction. (More mixing means more chance of an enzyme substrate collision). In future attempts a different range of pHs could be used (for example: 4.1, 4.2, 4.3, 4.4... instead of 5.9 then 7.0) so that it would help confirm a stronger trend as there would be more points to plot on the graph. Going beyond the pH of 8.8 would show if the optimum was really pH 8.8 or if the rate of reaction did increase on other pHs beyond 8.8 and when the enzyme actually becomes denatured. Also investigating how different factors affect the rate of reaction with the enzymes like the change of temperature or the concentration (instead of 2% protease) would be considered to illustrate the characteristics of enzymes. However all the aspects mentioned above could have affected the results but a general trend was discovered therefore showing that they really did not have a huge affect. ?? ?? ?? ?? Page 1 ...read more.

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