Km And Vmax Lab Report

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Determining the of value of Km & Vmax at 37°C and 65°C using Michaelis-Menten

Introduction
Biological reactions involves the use of enzymes, which acts as catalyst. Reactions only occur if the substrate fits into the active site of an enzyme and so changes in temperature can denature the enzyme and therefore makes it void[1]. Trypsin is a digestive enzyme used to breakdown proteins such as BAPNA in the body, It has a optimum temperature of 37 °C[2]. The aim of this experiment is to calculate Km and Vmax at 37 and 65 °C by using Michaelis-Menten and Eadie-Hofstee Graph in order to determine how temperature affects the rate of digestion of BAPNA by trypsin.
Method
Eight ml of six different substrate concentrations (2000, 1500, 1250, 1000,
Figure 1: Michaelis-Menten graph used to estimate the Km and Vmax at 37 & 65 °C Table 3: showing the values of Km and Vmax calculated from graphpad
Km and Vmax For Michaelis-Menten From Graphpad at 37 And 65°C

37°C
65°C
Km
6284
6106
Vmax
0.2585 0.14 00

Table 4: Shows the values used to produce a Eadie-Hofstee graph at 65°C

Initial Velocity/[S] Against Initial Velocity Concentration At 37°C
Initial velocity V0 / [S] x 10-5( µMmin-1dm3mol-1)

Initial velocity V0 (µMmin-1)
3.4821
0.03980
3.6215
0.03104
3.7085
0.02649
3.7102
0.02120
4.0357
0.01153

Figure 2: Eadie-Hofstee linear graph to determine Km and Vmax at 37°C

Table 5: shows the values used to produce a Eadie-Hofstee graph at 65°C
Initial Velocity/[S] Against Initial Velocity Concentration At 65°C
Initial velocity V0 / [S] x 10-6 (µMmin-1dm3mol-1)
Initial velocity V0 (µMmin-1)
3.8688
0.004422
3.7335
0.003200
4.4491
0.003178
4.3945
0.002511
3.9657
0.001133

Figure 3: Eadie-Hofstee linear graph to determine Km and Vmax 65°C

Km and Vmax For Eadie-Hofstee Kinetic Graph at 37 And