Isolation Of Polyphenol Oxidase Biology Essay

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INTRODUCTION

Enzymes are biological, globular molecules that catalyze or speed up a certain reaction. They cause a specific chemical change in all body parts, one example is the breaking down of the food we eat (1, 2). Enzymes are made up of amino acids, and these are proteins. Every enzyme has its own specific role and reaction to speed up depending on its property (3). Enzyme Kinetics is the process wherein the rate of chemical reaction catalysed by an enzyme is measured.

E + P

ES

E + S

Figure 1. General Equation for Enzyme-Catalyzed Reaction

As seen in Figure 1, a substrate (S) binds to an enzyme (E) making up the ES complex. Afterwards, dissociation of ES complex to the product (P) and recycled enzyme (E) happens.

MATERIALS AND METHODS

The experiment consists of 3 parts. First is the isolation of Polyphenol Oxidase wherein a 15.0g sample of potato was peeled and finely chopped. After homogenizing it with phosphate buffer in a blender, the extract was filtered using cheesecloth and was collected in a beaker. Second part is the Enzyme Kinetics of Uninhibited Reaction wherein 5 test tubes were prepared with assigned volumes of catechol and phosphate buffer. 0.20 ml of the extracted enzyme was then added in every test tube before immediately reading the absorbance at 420 nm every 5 seconds interval. For the last part, Enzyme Inhibition, 5 test tubes same as the second part was prepared but this instance, ascorbic acid was added and the same procedure for every test tube was done. For a more detailed procedure, refer to Figure 2.

Isolation of Polyphenol Oxidase

Cover w/ aluminum foil

Phosphate Buffer

Ice bath

+ 40 ml phosphate buffer

Ice bath

Filter

15 g potato peeled and chopped

Enzyme Kinetics of Uninhibited Reaction

Test Tube

0.100M Catechol

0.200M Phosphate Buffer

1

0.20 ml

2.6 ml

2

0.40 ml

2.4 ml

3

0.80 ml

2.0 ml

4

1.60 ml

1.20 ml

5

2.00 ml

0.60 ml

Table 1. Designated amount of Catechol and Phosphate buffer per test tube

*Read absorbance at 420 nm every 5 sec

Tube 1

* Repeat for every test tube

Enzyme Inhibition

Test Tube

0.100M Catechol

0.100M Ascorbic acid

0.200M Phosphate Buffer

1

0.20 ml

0.10 ml

2.5 ml

2

0.40 ml

0.10 ml

2.3 ml

3

0.80 ml

0.10 ml

1.9 ml

4

1.60 ml

0.10 ml

1.10 ml

5

2.00 ml

0.10 ml

0.70 ml

Table 2. Designated amount of reagents per test tube

* Repeat procedure in Part B

Figure 2. Schematic Diagram of Procedure

RESULTS AND DISCUSSIONS

The tabulated data and results of the absorbance at 420 nm for the Uninhibited and Inhibited reaction can be seen in Table 3 and 4.

Time (s)

Absorbance at 420 nm

Tube 1

Tube 2

Tube 3

Tube 4

0

0.746

0.676

0.700

0.480

5

0.773

0.731

0.788

0.795

10

0.798

0.772

0.845

0.867

15

0.820

0.806

0.889

0.914

20

0.837

0.828

0.923

0.955

25

0.861

0.843

0.946

0.986

30

0.877

0.854

0.959

1.012

35

0.877

0.861

0.964

1.032

40

0.899

0.866

0.962

1.044

45

0.910

0.868

0.957

1.049

50

0.912

0.868

0.947

1.042

55

0.899

0.867

0.937

1.029

60

0.870

0.864

0.926

1.011

Table 3. Absorbance at 420 nm for Uninhibited Reaction

In Table 3, the test for uninhibited reaction of the enzyme polyphenol oxidase was observed by tabulating the reading every 5 second interval.

Figure 3. Graph of absorbance for Uninhibited reaction

In Figure 3, the graphed form of data tabulated in Table 3 from time 10 up to 35, or the point wherein the lines ascend were plotted. Every line has its own designated equation bearing the slope and its y-intercept. The slope of every equation stands for the velocity of every reaction in every test tube.

Time (s)

Absorbance at 420 nm

Tube 1

Tube 2

Tube 3

Tube 4

0

0.23

0.224

0.28

0.341

5

0.23

0.224

0.279

0.34

10

0.231

0.229

0.278

0.34

15

0.26

0.26

0.279

0.34

20

0.304

0.29

0.28

0.341

25

0.347

0.302

0.283

0.343

30

0.385

0.336

0.311

0.346

35

0.417

0.393

0.356

0.424

40

0.443

0.421

0.397

0.472

45

0.462

0.43

0.433

0.509

50

0.474

0.452

0.461

0.546

55

0.485

0.482

0.481

0.573

60

0.496

0.497

0.499

0.602

Table 4. Absorbance at 420 nm for Inhibited Reaction

Just like in Table 3, the reading every 5 second interval was tabulated for every test tube.

Figure 4. Graph of absorbance for Inhibited Reaction

Reaction System

Velocity (mM/s)

Tube 1

Tube 2

Tube 3

Tube 4

Uninhibited

3.4

3.5

4.7

6.6

Inhibited

5.5

5.6

6.5

8.1

Table 5. Velocity of enzyme in Uninhibited and Inhibited reaction

The velocity tabulated in Table 5 was obtained by looking at the slope of every equation in the graphs plotted. Every slope value was multiplied by 1000 to convert it to mM unit.

Tube #

1/[S], (mM-1)

1/V, (s/mM)

Uninhibited Reaction

Ascorbic Acid inhibited

1

0.1499

0.294

0.182

2

0.075

0.286

0.179

3

0.0375

0.213

0.154

4

0.0188

0.152

0.123

5

0.015

0.333

0.152

Table 6. Lineweaver-Burke Analysis

The reciprocal of the substrate concentration computed by using the dilution formula M1V1 = M2V2 was tabulated in Table 6 together with the reciprocal of the velocity both in uninhibited and inhibited reaction.

Figure 5. Lineweaver-Burke Graph

Parameters

Uninhibited

Ascorbic Acid inhibited

Vmax

4.37

7.26

KM

1.97

2.48

Mode of Inhibition: Uncompetitive Inhibition

The graph in Figure 5 shows that the two lines are parallel with each other demonstrating an uncompetitive inhibition.

Figure 6. Oxidation of Catechol to Benzoquinone

Figure 7. Structure of Ascorbic acid

Figure 8. Mechanism of Ascorbic Acid as an antioxidant

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