Biochem Enzyme

Universiti Tunku Abdul Rahman (Kampar Campus) Faculty of Science, Engineering, and Technology Bachelor of Science (Hons) Biotechnology Year 2 Semester 1 UESB 2142 Laboratory 2A (II) Biochemistry and Cell Metabolism Lecturer: Mr. James Wee Sheau Yang Student’s Name: Cheah Hong Leong Student’s ID: 08AIB03788 Experiment No. 3 and 4 Title: Isolation of the Enzyme Peroxidase from Radish and Properties of Peroxidase. Date: July 13, 2009

Title: Isolation of Peroxidase from Radish and Properties of Peroxidase

Objectives: –

To extract the enzyme Peroxidase from the radish.

–

To determine the effect of concentration of enzyme Peroxidase on the rate of reaction based on the optical density obtained through spectrophotometer.

–

To compare the rate of reaction catalyzed by crude Peroxidase with that of the commercial Peroxidase.

Results: Table 1: Optical Density Readings over Time Obtained from Spectrophotometer on Five Test Tubes Time, s

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150

Optical Density, A Test tube

Test tube

Test tube

Test tube

Test tube

1

2

3

4

5

0.265 0.275 0.285 0.312 0.325 0.340 0.358 0.372 0.387 0.398 0.410 0.421 0.436 0.449 0.461 0.478 0.490 0.501 0.508 0.519 0.528 0.537 0.549 0.560 0.568 0.576 0.584 0.593 0.601 0.608

0.273 0.340 0.414 0.470 0.503 0.520 0.535 0.546 0.558 0.572 0.584 0.597 0.610 0.624 0.636 0.648 0.661 0.674 0.685 0.698 0.709 0.721 0.732 0.742 0.754 0.763 0.775 0.783 0.792 0.800

0.145 0.176 0.208 0.242 0.279 0.311 0.347 0.380 0.412 0.442 0.478 0.502 0.526 0.554 0.582 0.604 0.630 0.650 0.670 0.687 0.703 0.719 0.734 0.748 0.760 0.770 0.780 0.789 0.800 0.806

0.119 0.120 0.121 0.122 0.123 0.125 0.126 0.127 0.128 0.128 0.129 0.130 0.131 0.132 0.133 0.133 0.134 0.134 0.135 0.136 0.136 0.137 0.137 0.138 0.138 0.139 0.139 0.140 0.140 0.140

0.100 0.126 0.147 0.170 0.191 0.214 0.231 0.250 0.274 0.286 0.300 0.318 0.333 0.349 0.360 0.374 0.385 0.395 0.408 0.419 0.429 0.440 0.450 0.460 0.466 0.474 0.481 0.489 0.498 0.505

155 0.615 0.808 0.814 0.141 0.515 160 0.623 0.814 0.820 0.141 0.522 165 0.630 0.822 0.827 0.141 0.528 170 0.636 0.828 0.833 0.141 0.533 175 0.642 0.834 0.839 0.142 0.537 180 0.648 0.839 0.843 0.142 0.542 *The contents in the test tube from 1 to 5 were refers to Laboratory Manual UESB 2142 Laboratory 2A (I) Biochemistry and Cell Metabolism page 7. *Graph 1 to 5 in attachments were plotted based on the table above and the slope of the graphs were calculated to obtain the zero order reaction rates. *Optical density does not have true unit; Absorbance, A is used.

Zero order reaction rate calculation: Graph 1: Optical Density versus Time for Test Tube 1 Gradient of the graph = 0.23A/84s = 2.74 x 10-3As-1 Zero order reaction rate = 2.74 x 10-3 As-1 Graph 2: Optical Density versus Time for Test Tube 2 Gradient of the graph = 0.2A/78s = 2.56 x 10-3As-1 Zero order reaction rate = 2.56 x 10-3 As-1 Graph 3: Optical Density versus Time for Test Tube 3 Gradient of the graph = 0.31A/46s = 6.74 x 10-3As-1 Zero order reaction rate = 6.74 x 10-3 As-1 Graph 4: Optical Density versus Time for Test Tube 4 Gradient of graph = 0.01A/55s = 1.82 x 10-4As-1 Zero order reaction rate = 1.82 x 10-4 As-1 Graph 5: Optical Density versus Time for Test Tube 5 Gradient of graph = 0.34A/76s

= 4.47 x 10-3 As-1 Zero order reaction rate = 4.47 x 10-3 As-1 Calculation on concentration of Peroxidase: Weight of dried crude Peroxidase = 0.0204g = 20.4mg Volume of distilled water added = 10ml Concentration of crude Peroxidase = 20.4mg/10ml = 2.04mg/ml Test tube 1- M1V1 = M2V2 (2.04mg/ml)(2.0ml) = M2 (3.2ml) M2 = 1.275mg/ml Test tube 2- M1V1 = M2V2 (2.04mg/ml)(1.0ml) = M2 (3.2ml) M2 = 0.638mg/ml Test tube 3- M1V1 = M2V2 (2.04mg/ml)(0.5ml) = M2 (3.2ml) M2 = 0.319mg/ml Concentration of commercial Peroxidase = 10mg/100ml = 0.1mg/ml Test tube 5- M1V1 = M2V2 (0.1mg/ml)(1.0ml) = M2 (3.2ml) M2 = 0.031mg/ml

Table 2: The Zero Order Reaction Rate and the Concentration of Enzyme Peroxidase Test

Peroxidase concentration, Zero Order Reaction Rate, x

Tube 1 2 3

mg/ml

10-3 As-1 1.275 0.638 0.319

2.74 2.56 6.74

4 *5

0.000 0.031

0.18 4.47

*The Peroxidase used in test tube 5 was commercial Peroxidase. *Graph 6 in attachment was plotted based on the above table.

Conclusion and Discussion: By theory, the zero order reaction rates between hydrogen peroxide and pphenylenediamine should be directly related to the concentration of peroxide that catalyzes the reaction. However from Graph 3, the zero order reaction rate obtained from Test tube 3 was extremely highest among the Test Tube 1, 2, and 3 even though the concentration of the peroxide in Test Tube 3 was the lowest. This indicates that some error(s) had occurred when the experiment was in progress. However, since all the crude preparation of diluted Peroxidase solution in the three test tubes were taken from the same source of crude Peroxidase solution, possibility of error in Experiment 3 was eliminated. The only possible errors might happen only in Experiment 4. One of the possible errors might be the contamination of cuvette by Peroxidase from Test tube 1 and 2. The structure of cuvette tends to trap some solution inside it even after rinsed with distilled water. Additional of Peroxidase in little amount in cuvette can cause high increase in reaction rate as enzyme is only needed in little quantity.

When comparing the crude peroxide in Test Tube 1 and 2, the Peroxidase concentration in Test tube 1 was two-fold than that of in Test Tube 2. However, the zero order reaction rate calculated in Test Tube 1 was only slightly higher than that of Test Tube 2. This might cause by the limitation effect of the concentration of either hydrogen peroxide or p-phenylenediamine or both of them. Peroxidase might be needed in little amount to catalyze the reaction, over excess of quantity of Peroxidase did not further increased the reaction rate.

From Graph 4, little increase in optical density obtained from the spectrophotometer indicated that there was still has reaction of hydrogen peroxide and p-phenylenediamine, even though lack with Peroxidase. One possible explanation was the contamination or presence of little amount of inorganic catalyst in water or any other solutions being added together into the test tube. The inorganic catalyst presence in little amount can be metallic ions such as copper ion, Cu2+, manganese dioxide, and silver ion. From the Graph 5 and Graph 6, the zero order reaction rate catalyzed by commercial Peroxidase was much higher than that of crude preparation of Peroxidase. From Graph 6, the concentration of commercial Peroxidase was only 0.031mg/ml, which was much lower than crude Peroxidase concentration in Test Tube 1, 2, and 3. However, the zero order reaction rate in Test Tube 5 was much higher than that in Test Tube 1 and 2. One possible explanation for this result was the impurities of crude preparation of Peroxidase in Experiment 3. Although the concentration of crude enzyme in Test Tube 1 and 2 seemed to be higher than concentration of commercial enzyme in Test Tube 5, however, any possible improper techniques used in extraction of Peroxidase from radish might cause impurities in the extracted enzyme solution. The extracted enzyme might contain largely of other compounds presence in the radish cells and only little amount of Peroxidase. Another possible reason was the preparation techniques in commercial industry were much sophisticated. References: Campbell, N.A. & Reece, J.B., 2005. Biology. 7th ed. CA: Pearson Benjamin Cummings. Voet, D.J., Voet, J.G., & Pratt, C.W., 2008. Principles of Biochemistry. 3rd ed. NJ: John Wiley & Sons, Inc. Wikipedia 2009, Hydrogen Peroxide, online, retrieved 25 July 2009, from http://en.wikipedia.org/wiki/Hydrogen_peroxide Wikipedia

2009,

Peroxidase,

online,

http://en.wikipedia.org/wiki/Peroxidase

retrieved

25

July

2009,

from

Attachments: Graph 1: Optical Density versus Time for Test Tube 1 Graph 2: Optical Density versus Time for Test Tube 2 Graph 3: Optical Density versus Time for Test Tube 3 Graph 4: Optical Density versus Time for Test Tube 4 Graph 5: Optical Density versus Time for Test Tube 5 Graph 6: Zero Order Reaction Rate versus Peroxidase Concentration