The effect of variable catechol concentration on the rate of enzyme activity

Niloufar Kosari

Lab Partners: Weiwei Liu, Ashkaan

Lab Section: 1219

Date: June/2/2013

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Introduction

Enzymes are biological molecules that are involved in chemical interconversions in living organisms. They act as catalysts that greatly accelerate both the rate and specificity of metabolic reactions(Garrette & Grisham, 1999). In our experiment a number of tests were carried out on a specified enzyme with the main aim of the experiment being to demonstrate some properties of enzymes. In the experiments, the activity of the enzyme polyphenol-oxidase from potato extract was measured under varying concentration of substrate catechol at Ph 6. The question was to find out what effect concentration of catechol on the rate of polyphenol-oxidase from potato extract reaction.

The variable under study in this experiment was the substrate concentration. In order to show its effect on enzyme activity, the concentration of the substrate used (catechol) was increased in the following proportions: 0.2 ml, 0.5ml, 1 ml, 2.5 ml and 3 ml. With this in mind the following hypothesis was made; as the substrate concentration is increased, the rate of reaction increases too. This increase is observed up to the point where all available enzymes are occupied by the substrate. In this case, this is the maximum rate and it can also be referred to as the plateau. This hypothesis is a reflection of what is expected from the experiment. However, an understanding of the occurrence of the phenomena was necessary and it is as presented in the following section.

In the world of chemistry, most of enzymes are proteins. Each particular enzyme has its own unique physical structure that is vital for its function. The importance of this shape is that it “fits” into the shape of the reacting molecule or molecules in a reaction where the enzyme acts as a catalyst. The enzyme “fit” enables reacting molecules to bond together at the appropriate bonding sites and this “lowers” the activation energy of the chemical reaction: which is the purpose of the enzyme (Liljas, Liljas, Piskur, Nissen, Kjeldgaard, & Lindblom, 2009).

In this experiment a phenolic compound, Catechol, naturally found in many plants (potatoes and apples) was used. Injured tissues of such plants release catechol, which is rapidly oxidized by their own catechol-oxidase to benzoquinone, that spontaneously get converted to the brown pigment Melanin (Creveling, 2000).

In this case, the catechol was reacted with polyphenol oxidase in order to measure the accumulation of colored products (the change in absorbance) in a spectrophotometer at 475 nm. The reaction for this process is as shown in the figure below:

 

 

This chemical reaction shows the conversion of catechol to benzoquinone in the presence of the enzyme, polyphenol oxidase (Creveling, 2000). This is the chemical reaction that will be expercted to be occuring during the experiment.

The outcome of the experiment was then used to carry out various analyses as required for this exercise. During the experiments, the change in color of the catechol was observed to take place. In addition, the graphical analysis done on the numerical results obtained from the experiment brought out the expected outcomes from such an experiment. The hypothesis posted earlier was also proven.

Method

In this experiment, the following chemicals and apparatus were used; potato extract containing the enzyme polyphenol-oxidase, 0.1% Catechol (substrate), Phosphate buffer of pH 6.0, test tubes, 1 mL, 5 mL and 10 mL pipet, two 100 mL beakers, timer and spectrophotometer. A five-milliliter reaction mixture containing each of the named chemicals was prepared in a test tube. The mixture was mixed rapidly as it was poured into a cuvette. The timer was started as soon as the substrate and the enzyme were both present in the mixture. Then, the absorbance was measured by the spectrophotometer at a wavelength of 475 nm every 30 seconds.

Six samples with various concentration of Catechol were made according to the following Table:

Sample

1

2

3

4

5

6

Substrate (mL)

0

0.2

0.5

1

2.5

3

Buffer (mL)

4

3.8

3.5

3

1.5

1

Enzyme (mL)

1

1

1

1

1

1

Total Volume (mL)

5

5

5

5

5

5

 

While doing the precise measurements, the potato extract was added in the mixture last and the recording of time stated at that instance. The readings of the absorbance of each tube were then recorded for increasing time intervals up to the 20-minute mark.

Results

In this section, the results obtained from each experiment are presented. The data was then used to carry out various analyses in graphical means as shown in the diagrams following the table of results.

Table 1: Absorbance values for produced Melanin at six various substrate concentrations. This is the raw data that was used to plot the graphs of absorbance against time and compared with the hypothesis in the beginning of this report.

Time (min)

Absorbance at 475 nm

1

2

3

4

5

6

0.3

0.105

0.118

0.0576

0.078

0.198

0.132

1

0.115

0.097

0.127

0.112

0.212

0.183

1.3

0.094

0.152

0.189

0.124

0.228

0.223

2

0.126

0.159

0.221

0.196

0.289

0.271

2.3

0.169

0.179

0.183

0.177

0.318

0.303

3

0.163

0.214

0.22

0.186

0.346

0.36

3.3

0.127

0.211

0.188

0.189

0.415

0.414

4

0.135

0.258

0.171

0.223

0.442

0.436

4.3

0.135

0.231

0.221

0.217

0.476

0.515

5

0.134

0.271

0.214

0.22

0.493

0.53

5.3

0.118

0.298

0.252

0.224

0.515

0.544

6

0.169

0.305

0.172

0.275

0.533

0.566

6.3

0.154

0.341

0.256

0.287

0.615

0.626

7

0.19

0.344

0.225

0.288

0.599

0.613

7.3

0.157

0.38

0.241

0.515

0.651

0.622

8

0.157

0.41

0.285

0.3

0.66

0.66

8.3

0.154

0.428

0.301

0.362

0.704

0.68

9

0.147

0.474

0.308

0.385

0.704

0.682

9.3

0.16

0.474

0.298

0.371

0.712

0.677

10

0.142

0.482

0.315

0.37

0.672

0.682

10.3

0.14

0.497

0.373

0.402

0.718

0.657

11

0.14

0.511

0.398

0.413

0.707

0.698

11.3

0.17

0.535

0.38

0.417

0.718

0.709

12

0.162

0.583

0.402

0.426

0.701

0.672

12.3

0.138

0.625

0.389

0.44

0.712

0.68

13

0.157

0.585

0.363

0.447

0.738

0.726

13.3

0.181

0.594

0.377

0.481

0.732

0.715

14

0.167

0.613

0.391

0.462

0.769

0.696

14.3

0.165

0.64

0.385

0.555

0.789

0.709

15

0.161

0.65

0.398

0.531

0.741

0.693

15.3

 

0.671

0.47

0.506

0.766

 

16

 

0.69

0.447

0.549

0.763

 

16.3

 

0.671

0.46

0.531

0.772

 

17

 

0.682

0.442

0.568

0.766

 

17.3

 

0.714

0.44

0.606

0.766

 

18

 

0.69

0.497

0.563

0.782

 

18.3

 

0.679

0.506

0.578

0.779

 

19

 

0.666

0.44

0.601

0.769

 

19.3

 

0.751

0.515

0.568

0.789

 

20

 

0.764

0.504

0.601

0.782

 

 

From this data, the following graphs were obtained:

 

Figure 1: Melanin production using six different concentration of Catechol for 20 minutes. The graph depicts the progression of enzymatic reaction for different substrate concentrations and is a reflection the Melanin production. The Melanin production increases over 20 minutes. The trend lines were assigned. The equation and the R2 of the curves of 0mL, 0.2mL, 0.5mL, 1mL, 2.5mL and 3mL are y = -0.000x2 + 0.007x + 0.111, R2 = 0.954, y = -0.000x2 + 0.052x + 0.053, R2 = 0.987, y = -0.000x2 + 0.023x + 0.111, R2 = 0.933, y = -0.000x2 + 0.034x + 0.078, R2 = 0.982, y = -0.002x2 + 0.078x + 0.155, R2 = 0.978, y = -0.004x2 + 0.106x + 0.0.087, R2 = 0.986, respectively.

From figure one, the following table of the rate of melanin production for each sample was obtained.

Table 2: Rate of melanin production for each sample of substrate concentration: This is the information that was then used to plot the plot of melanin production level against concentration as shown in figure two.

 

Sample

Substrate concentration (mL)

Rate of melanin production over 2 to 5 minutes

1

0

-0.005

2

0.2

0.034

3

0.5

0

4

1

0.013

5

2.5

0.073

6

3

0.091

 

 

 

Figure 2: kinetics of the enzyme reaction measured by the rate of the production of Melanin through the absorbance values of six different concentrations of Catechol over 2-5 minutes. The rate of Melanin production except for the 0.2 mL Catechol concentration between 2-5 minutes increases as the substrate concentration increases. The curve equation is y = 0.0099x2 – 0.0011x + 0.0084 and the R² = 0.95443

 

 

Discussion

From figure one, the shape of the figures can be explained by the fact that the rate of melanin production is affected by more than just the concentration of the substrate. That is, the rate of melanin production could have been affected by factors other than catechol oxidase activity, such as the intracellular organization, pH concentration and temperature. However, the concentration in this experiment was the effect of substrate concentration. From a cellular perspective, it can be seen that the activity of melanin formation depended on the amount of the enzyme in solution. For instance, in the solution without the enzyme the formation of melanin was so slow that it appeared to be inactive as compared to the solutions with enzymes. The reaction with the highest amounts of the enzyme was the most prolific. Then, from a molecular perspective the shapes of the graphs can be explained as follows: As catechol was oxidized by the enzyme to form 0-benzoquinone this was followed by the formation of a heterogeneous group of polymers called melanin. As the polymers got larger, their colors were seen to deepen from pink-gold through orange-brown and finally to an intense brown-black color. Since the larger molecules are less soluble in water they would eventually precipitate from the solution. The process begins as a spontaneous activity as shown in the initial stages of the graphs in figure one which then slows down as melanin formation comes to an end.

As earlier stated in the hypothesis, as the concentration of the substrate was increased, the rate of reaction increased as well (with exception of the second point.) The increase in enzyme activity for each level of substrate concentration was observed up to a point where the relationship became horizontal. From the theory on enzyme activity, this is the point where the enzyme is occupied by the substrate. In this case, this is the maximum rate and it can also be referred to as the plateau. The Melanin production in each salt concentration increases over the time. This is because the reaction proceeds as the time passes.

Despite the expected outcome being reflected by the experimental results, some errors were noted. For instance, the data produced irregular graphs that could not be conclusively used for analysis. This then prompted the use of interpolation techniques in order to carry out the required analysis. In addition, it was observed that the reaction rate for a substrate concentration on 0.2ml was higher than that of 0.5ml and 1 ml of concentration. This is also an experimental error since reaction rates should increase progressively with increase in substrate concentration. However, this error was not consistent since the other substrate concentrations followed the expected trend. These errors might have come about due to human related errors such parallax errors when measuring the various fluid concentrations and improper timing when using the timer or apparatus inherent errors such equipment wear and tear due to aging. However, since the experiment yielded the expected outcomes, it can be considered to have been a successful exercise.

 

 

 

References

Creveling, C. R. (2000). Role of Catechol Quinone Species in Cellular Toxicity. Tennessee: F P Graham Company.

Garrette, R. H., & Grisham, C. M. (1999). Biochemistry. Philadelphia: Saunders College Publishers.

Liljas, A., Liljas, L., Piskur, J., Nissen, P., Kjeldgaard, M., & Lindblom, G. (2009). Textbook Of Structural Biology. Singapore: World Scientific Publishing Company.

Logan, R. 2003. Enzymatic Reactions. Biology 21 Lab Manual. Santa Monica College.

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