concentration were increased, then the reaction would occur faster since the enzyme would be more quickly and easily bounded by the substrate. Thus, the solution containing the most amount of substrate should have the greatest reaction rate. However, this does not hold true. The slopes of the lines for the first four solutions increase (.0003 mM/min, .0005 mM/min, .0010 mM/min, .0012 mM/min, respectively). The fifth solution has a slope of .0010, lower than that of the fourth solution. It is important to keep in mind, that error could have been present during this experiment. However, since the results did not match the state hypothesis, it would be critical to replicate the experiment for further results and thus better analysis. Graph 6 exhibits the effect of substrate concentration on enzyme activity. The enzyme activity increased during the first four solutions. However, the enzyme activity decreased from .001260 micromoles p- nitrophenol/min/ml in the fourth solution to .001070 micromoles p-nitrophenol/min/mL in the fifth solution. Although the enzyme activity dropping could be an error, it could also be explained by the fact that there are a limited number of active sites. Therefore, excess substrate would not be able to bind to the enzyme. This can also be explained by how close the enzyme activity was in the fourth and fifth solution. Graph 7 is a Line Weaver-Burk plot that also related to this part of the lab. The trend line in this graph gives us the equation: 1/V 0 = 2659.3(1/S) + 288.29. The Michaelis-Meneten constant measures the substrate concentration at half of the maximum velocity. The x-intercept, which can be found by setting 1/V 0 equal to 0, is found to be -.1084. Because the Michaelis-Menten constant, K m , is equal to -1 divided by the x-intercept, it is found to be 9.225. On the other hand, the y-intercept can be found by setting 1/S equal to 0. The y-intercept also equals 1/V max , which is found to be .0035. When comparing this V max value to the V max found from Graph 5, it is seen that the reaction was not completed to its fullest; instead, it was only a fourth complete. From a numerical sense, the V max obtained from Graph 5 was .0013 According to Table 11, another hypothesis was confirmed to be correct. When more enzyme was added into solution, the value of the OD increased. From tube 1a to 2a, the OD increased from .236 to .433. The “better blank” would be the serum with the active enzyme since the spectrophotometer would be accustomed to read with active enzymes present. Thus, the
spectrophotometer would be able to read all solutions containing the active enzymes with more accuracy. If the blank containing the boiled enzyme was used, it would not give as accurate readings cine the enzyme would be inactivated due to its denatured feature. The tertiary structure of the enzyme would be broken. Furthermore, the concentration of the buffer was not the same in all the samples. Because the buffer is used to maintain the pH of the enzyme, so it is at its optimal level, the results could be affected.
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