Part B: Change the amount of enzyme
Table 4: The absorbances of 5 cuvettes that measured at 475nm during a given time interval using spectrophotometer.
Time (s) Absorbance @ 475nm Cuvette 1 Cuvette 2 Cuvette 3 Cuvette 4 Cuvette 5
0 0.003 0.003 0.004 0.002 0.009
30 0.003 0.004 0.008 0.013 0.021
60 0.005 0.005 0.013 0.016 0.031
90 0.005 0.008 0.019 0.020 0.040
120 0.007 0.011 0.022 0.033 0.051
Figure 4: The graph of time vs. absorbance of 5 cuvettes that contained different amount of enzyme.
Part C: Change the amount of substrate
Table 5: The absorbances of 5 cuvettes that measured at 475nm during a given time interval using spectrophotometer
Time (s) Absorbance @ 475nm Cuvette 1 Cuvette 2 Cuvette 3 Cuvette 4 Cuvette …show more content…
In a case where the enzyme concentration is kept constant and the concentration of substrate is increased, the velocity of the reaction will increase rapidly until half of the enzyme becomes saturated with substrate (refer to figure 5). Also, analyzing the graphs suggest that the inhibition type of Cinnamic acid was noncompetitive inhibitor of tyrosinase’s reaction using the Michaelis-Menten and Lineweaver-Burk plots as expectation. As you can see in figure 7, which is Michaelis-Menten plot that showed the “without inhibitor’ and “with inhibitor” curves and you can compared the 2 curve to see if it what type of inhibitor. The graph had 2 sigmoidal curves, 1 is ‘without inhibitor’ and 1 is ‘with inhibitor’. The curve of ‘without inhibitor’ was on top of the ‘with inhibitor’. Since the inhibitor is Cinnamic acid, which is a non-competitive inhibitor according to ncbi.nlm.nih.gov, the ‘with inhibitor’ will be lower and never reach the Vmax of the ‘without inhibitor’ curve. In figure 8, which is the Lineweaver-Burk plot, the graph had 2 linear lines with an equation in a form of y=mx+b. As you can see in the graph, the ‘with inhibitor’ (Cinnamic acid) line was on top of the ‘without inhibitor’. Since it is a non-competitive, the 2 lines did not intersect at y-axis. Also, Cinnamic acid is competitive inhibitor; therefore the Vmax is decrease (#1 Lehninger). This lab used the Lineweaver- Burk plot to calculate the Km and Vmax. The graph was obtained by taking the reciprocal of the Michaelis-Menten Kinetics equation, which give you a linear line (chemwiki.ucdavis.edu). The linear data is easier to read and easier to calculate Vmax, Km than the sigmoidal graph of Michaelis-Menten plot. Refer to table 9, the Vmax of ‘without inhibitor” was 3.335x104 μMoles/min and the Vmax of ‘with inhibitor’ was 2.921x10-4
Table 4: The absorbances of 5 cuvettes that measured at 475nm during a given time interval using spectrophotometer.
Time (s) Absorbance @ 475nm Cuvette 1 Cuvette 2 Cuvette 3 Cuvette 4 Cuvette 5
0 0.003 0.003 0.004 0.002 0.009
30 0.003 0.004 0.008 0.013 0.021
60 0.005 0.005 0.013 0.016 0.031
90 0.005 0.008 0.019 0.020 0.040
120 0.007 0.011 0.022 0.033 0.051
Figure 4: The graph of time vs. absorbance of 5 cuvettes that contained different amount of enzyme.
Part C: Change the amount of substrate
Table 5: The absorbances of 5 cuvettes that measured at 475nm during a given time interval using spectrophotometer
Time (s) Absorbance @ 475nm Cuvette 1 Cuvette 2 Cuvette 3 Cuvette 4 Cuvette …show more content…
In a case where the enzyme concentration is kept constant and the concentration of substrate is increased, the velocity of the reaction will increase rapidly until half of the enzyme becomes saturated with substrate (refer to figure 5). Also, analyzing the graphs suggest that the inhibition type of Cinnamic acid was noncompetitive inhibitor of tyrosinase’s reaction using the Michaelis-Menten and Lineweaver-Burk plots as expectation. As you can see in figure 7, which is Michaelis-Menten plot that showed the “without inhibitor’ and “with inhibitor” curves and you can compared the 2 curve to see if it what type of inhibitor. The graph had 2 sigmoidal curves, 1 is ‘without inhibitor’ and 1 is ‘with inhibitor’. The curve of ‘without inhibitor’ was on top of the ‘with inhibitor’. Since the inhibitor is Cinnamic acid, which is a non-competitive inhibitor according to ncbi.nlm.nih.gov, the ‘with inhibitor’ will be lower and never reach the Vmax of the ‘without inhibitor’ curve. In figure 8, which is the Lineweaver-Burk plot, the graph had 2 linear lines with an equation in a form of y=mx+b. As you can see in the graph, the ‘with inhibitor’ (Cinnamic acid) line was on top of the ‘without inhibitor’. Since it is a non-competitive, the 2 lines did not intersect at y-axis. Also, Cinnamic acid is competitive inhibitor; therefore the Vmax is decrease (#1 Lehninger). This lab used the Lineweaver- Burk plot to calculate the Km and Vmax. The graph was obtained by taking the reciprocal of the Michaelis-Menten Kinetics equation, which give you a linear line (chemwiki.ucdavis.edu). The linear data is easier to read and easier to calculate Vmax, Km than the sigmoidal graph of Michaelis-Menten plot. Refer to table 9, the Vmax of ‘without inhibitor” was 3.335x104 μMoles/min and the Vmax of ‘with inhibitor’ was 2.921x10-4