The absorbance measured using standard samples were as shown in Table 1 below.
The absorbance measured in determining trypsin rate of activity in the absence of inhibitor was recorded in Table 2 below.
The absorbance measured in the determination of trypsin in the presence of inhibitor was recorded in Table 3 below.
The results of absorbance of the different standard samples were used to plot a graph of absorbance against p-nitroaniline concentration as shown in Figure 1 below.
Figure 1: A standard curve on the relationship between absorbance and p-nitroaniline concentration
Figure 2: Relationship between absorbance and p-nitroaniline concentration
Figure 3: Relationship between rate of reaction and p-nitroaniline concentration in the presence of an inhibitor
Reciprocal of the data used to plot Figure 2 and 3 was calculated and used to plot Figure 4 and 5 respectively.
Figure 4: Lineweaver-Burk plot in the absence of inhibitor
Determination of Vmax and Km was conducted as follows:
y = -302.55x + 514.35
1V = -302.55(1S) + 514.35
At Vmax, 1/[S] = 0
1[S]=0
y=514.35
1Vmax=514.35
Vmax=1514.35
Vmax=0.002
Km can be calculated where the line intercepts the x-axis (1/Km)
y=0
0= -302.55x +514.35
1Km=302.55 514.35
Km=1.7
Figure 5: Lineweaver-Burk plot in the presence of inhibitor
Determination of Vmax and Km was conducted as follows:
y = 6.8995x +41.409
1V = 6.8995(1S) + 41.409
At Vmax, 1/[S] = 0
1[S]=0
y=41.409
1Vmax=41.409
Vmax=141.409
Vmax=0.024
Km can be calculated where the line intercepts the x-axis (1/Km)
y=0
0= 6.8995x +41.409
1Km=-41.4096.8995
Km=-0.167
The values did not a clear indication of the mechanism of inhibition used by trypsin inhibitor. The noncompetitive inhibitors have Km value that is similar to a reaction with no inhibitor while competitive inhibitors have same Vmax. Uncompetitive inhibitors, on the other hand, cause a decrease in Vmax and Km values. There would be an increase in the rate of reaction resulting from the increased substrate concentration resulting in a reduced effect of the inhibitor. Addition of solutions to the cuvette should follow a particular order which ends with the addition of enzyme to prevent the reaction from starting earlier than expected. Increasing the temperature would have increased the rate at which the reactions took place compared to cold environments. The main advantage of a Lineweaver-Burk plot is that it is linear in nature and allows the determination of Km and Vmax and consequently the determination of the nature of inhibitors. The purpose of the standard curve was to enable determination of the concentration of p-nitroaniline. The curve enabled the conversion of absorbance of p-nitroaniline into concentration.
