Enzyme Kinetics

Gladys Ericka Galang Rae Angelei Regalado Group 6

May 5, 2014 May 5, 2014

Experiment 7 Catalytic Effect of Polyphenol Oxidase on Different Cathecol Concentrations Results and Discussions

Polyphenol oxidase was extracted from 250 g of banana by blending and addition of 0.1 M phosphate buffer pH 7. The crude extract was filtered and centrifuged to obtain the polyphenol oxidase used in the experiment. The bananas were placed in ice baths during the period of extraction to prevent oxidation of the enzyme.

The following graph is the plot of the absorbance of the solutions with different cathecol concentrations labelled A-D respectively. 0.7 0.6

Absorbance

This experiment aimed to study the kinetic activity of polyphenol oxidase extracted from banana on catechol solution and to assess the type of inhibition of benzoic acid on the enzyme.

0.5

A

0.4

B

0.3

C D

0.2 0.1

The same amounts of concentration of the catechol solution were used to measure the inhibitory effect of benzoic acid. To each of the solution, 1 mL of 0.3 mM benzoic acid was added. The absorbance of the solution was read at 420 nm against a fresh blank solution. UV-Vis spectrophometry used to measure the change in the absorbance of the reaction. Initially, the reactants have a specific absorbance that is read at 420 nm. As the reaction goes on and the substrate is consumed, there is a change of absorbance of the reaction. Beer-Lambert’s Law states that the change in absorbance of reactant or product is proportional to the change in concentration of that species during the reaction. (Boyer, 2000)

Time

10

110

210

Fig 1. Absorbance vs Time Plot without inhibitor

The following graph shows the plot of the absorbance versus the time of the solutions with the addition of benzoic acid as an inhibitor. 0.7 0.6 0.5 Absorbance

Four concentrations of cathecol solutions were prepared from the 10 mM stock solution: 4.8 mM, 1.2 mM, 0.6 mM and 0.3 mM. To each solution, 1 mL of the sample extract was added and was read at 420 nm against a blank solution of a mixture of 1 mL of the extract and 2 mL phosphate buffer.

0.4

A

0.3

B

0.2

C

0.1

D

0 Time

-0.1

10

110

210

Figure 2. Absorbance vs Time Plot with Inhibitor

The Michaelis-Menten was used to further analyse the data gathered.

The following table is the summary of the initial velocities of the reactions read, among other data calculated from the graph:

Conc Inhibited Uninhibited 4.8 0.0011 0.0011 1.2 0.0004 0.0002 0.6 0.0003 0.0001 0.3 0.0002 7x10^-5

BA

0 0

2

4

6

Vo

Figure 2. Michaelis-Menten graph The theoretical graph of the Michaelis-Menten equation is a sigmoidal graph. The graph from the experiment can help us conclude that the substrate concentration did not reach the maximum amount to reach the Vmax in the experiment. There are methods to linearize the MichaelisMenten equation for better quantification of the Vmax and Km. These are the Lineweaver-Burke equation, the Eadie-Hosftee plot and the Hanes-Woolf plot. The Lineweaver Burk equation is the graph of the velocity of the reaction versus the concentration of the substrate. This equation is derived using the double reciprocal of the Michaelis-Menten equation. (

)

BA

y = 1228.6x + 1079.9 R² = 0.9527

0 0

1

2

3

4

Figure 3. Lineweaver-Burk equation

0.0006

[S]

8000

1 / [S]

w/o BA

0.0002

w/o BA

10000

2000

y = 0.0002x + 0.0002 R² = 0.9983

y = 0.0002x - 4E-05 R² = 0.9952

y = 4187.2x + 1224.3 R² = 0.9458

4000

0.0012

0.0004

12000

6000

The initial velocity is the slope of the first lines plotted with a linear regression of 1. Plotting the initial velocity against the concentration of the substrate, we have the following graph:

0.0008

14000

1/V

Table 1. Summary of Data Calculated

0.001

16000

The Lineweaver-Burk plot of the transformed data is is one of the most influenced plots for studying the effects of inhibitors on enzymes with the easiest transformation. (http://academic.pgcc.edu/, 20) A disadvantage of this plot is that most experimental measurements involve relatively high [S] and are therefore crowded onto the left side of the graph. Furthermore, for small values of [S], small errors in Vo lead to large errors in 1/Vo and hence to large errors in Km and Vmax. (Voet, 2011) The graph of the uninhibited solutions gives us a K m value of 3.42 and a Vmax of 8.16x10-4 s-1. The graph of the inhibited solutions however gives us a Km 1.14 of and a Vmax of 9.26x10-4 s-. We cannot conclude the type of inhibition according to this graph because both the Vmax and the Km values rose. The Eadie-Hosftee equation is the plot of the Vo against the Vo/[S] given by the Michaelis-Menten equation rearranged into:

0.0007

7000

0.0006

6000

0.0005

5000 [S]/V

V

0.0004 0.0003

y = -0.3917x + 0.0006 R² = 0.6937

0.0002 0.0001

w/o BA

y = 572.3x + 1728.7 R² = 0.8914

BA

4000 3000 2000 1000

0

0

V/[S]

y = -198.73x + 5505.1 R² = 0.1831

0.0002 0.0004 0.0006 0.0008

0.001

0.0012

[S]

0 0

2

4

6

Figure 4. Eadie-Hostee plot of the uninhibited solutions

Figure 6. Hanes-Woolf graph uninhibited and inhibited solutions

This graph gives us a Km, which is the negative of the slope of the graph, of 0.3917 and a Vmax of 0.0006.

The Hanes-Woolf graph gives a line with a positive slope. The experimental data of the inhibited solutions however shows otherwise. The K m and Vmax values of the inhibited and the uninhibited solutions respectively are 1.74x10-3, -5.03x10-3 and 1x10-6 s-1, -9.14x10-7 s-1. The type on inhibition according to this graph is noncompetitive mixed.

0.00025

y = 0.0358x + 0.0002 R² = 0.2223

0.0002

of

both

the

V

0.00015 0.0001 0.00005 0 V/[S]

0

0.0002 0.0004 0.0006 0.0008

0.001

0.0012

Figure 5. Eadie-Hosftee graph of the inhibited solutions This graph of the inhibited solutions gives us a K m of -0.0358 and a Vmax of 0.0002. We can conclude that the type of inhibition according to this graph is uncompetitive inhibition. Ideally, the Eadie-Hosftee equation gives a line that is sloping downward. Errors might be encountered to result to a very different kind of graph. The Hanes-Woolf equation is the plot of [S]/v against [S] given by the equation: (

)

Considering the differences in the experimental and the theoretical values of the graphs and different conclusions with regards to the type of inhibition, it can be concluded that there are errors in the data collected, due to different factors such as errors in preparation of the sample, the oxidation of the sample, errors in placing the sample in the spectrophotometer, among others. References:  Voet, D. et al., 2011, Biochemistry, Courier/Kendallville  Boyer, R., 2000 Biochemistry, Benjamin Cummings  http://academic.pgcc.edu/~ssinex/excelets/enzyme _kinetics.pdf