Tyrosinase Report PDF

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Enzyme Kinetics: Tyrosinase
7 page lab report for the gen phys lab section...

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--BIOL 325 Lab Section: Thurs 1:00-3:50 Enzyme Kinetics: Tyrosinase Enzymes speed up reactions by lowering the activation energy of the reaction, and their kinetics can be measured in order to characterize the behavior of the enzyme. The purpose of this lab was to measure the activity of the tyrosinase enzyme. Tyrosinase has many other names such as monophenol monooxygenase, phenolase, monophenol oxidase, and cresolase. One of its most notable functions is its role in melanin production of converting dihydroxyphenylalanine (DOPA) into the intermediate dopaquinone. Dopaquinone is then spontaneously converted into dopachrome, which is a chromophore. The activity of the tyrosinase enzyme can be measured by observing either the disappearance of substrate or the appearance of product. Dopachrome, being a chromophore, has an absorbance maximum of 475 nm and can be used as a measure of tyrosinase activity in a spectrophotometer (Zhang). We study enzyme kinetics in terms of the following values: maximum velocity of the reaction (Vmax), the concentration of substrate that allows the enzyme to reach half of its maximal velocity (Km), concentration of substrate ([S]), concentration of enzyme ([E]), and time (t). In this lab, we set out to determine the extinction coefficient of dopachrome, obtain Km and Vmax values for tyrosinase, and measure the result of inhibiting tyrosinase activity with benzoic acid. We expected to observe typical Michaelis Menten kinetics when measuring tyrosinase activity. And, for the enzyme inhibition exercise, we hypothesized that benzoic acid would act as a competitive inhibitor of tyrosinase, as it structurally resembles the substrate for tyrosinase. Competitive inhibitors can be overcome by an increase in substrate concentration, so we expected to observe a higher Km but similar Vmax value when compared to the uninhibited tyrosinase kinetics. Previous studies have shown benzoic acid to work as a competitive inhibitor of mushroom tyrosinase (Loizzo 2012). Results: Serial dilutions were taken after a 15-minute incubation of DOPA and tyrosinase extract. A spectrophotometer was used to measure absorbance at a wavelength of 475 nm. Table 1 below shows that, as the concentration of DOPA increases, the absorbance values also increase as a result of higher enzyme activity. This is indicative of more dopachrome formation.

[Dopachrome], mM

Absorbance Abs/Con (mM^-1)

0 (blank)

0

0

0.125

0.006

0.000048

0.25

0.016

0.000064

0.5

0.018

0.000036

1

0.044

0.000044

2

0.088

0.000044

4

0.182

0.0000455

8

0.391

0.000048875

Table 1: Exercise 2, Absorbance of dopachrome solution measured at 475 nm.

After obtaining these values, we calculated the absorbance/concentration ratio for each tube in units of uM^-1, then averaged the ratios to obtain the molar extinction coefficient for dopachrome of 0.000041297 uM^-1. Table 2: Exercise 3, DOPA dilutions and absorbance (Abs) over time Time (s)

Abs at 0.5 mM

Abs at 1 mM

Abs at 2 mM

Abs at 4 mM

Abs at 8 mM

0

0

0

0.038

0.028

0.039

30

0

0

0.038

0.029

0.038

60

0.003

0

0.039

0.03

0.039

90

0.005

0

0.04

0.031

0.039

120

0.007

0

0.042

0.032

0.04

150

0.009

0

0.043

0.033

0.041

180

0.012

0

0.044

0.035

0.042

210

0.011

0.002

0.045

0.037

0.043

240

-

0.004

0.046

0.038

0.045

270

-

0.007

0.047

0.039

0.046

300

-

0.009

-

-

-

330

-

0.011

-

-

-

360

-

0.013

-

-

-

390

-

0.015

-

-

-

********We utilized Beer’s Law to calculate the concentration. A = Elc

Table 3: Exercise 3, DOPA dilutions and maximum concentration over time [DOPA]

Tmax (s)

Conc max

Conc initial

Delta Conc

0.5

210

266.36

0

266.36

1

390

363.22

0

363.22

2

270

1138.10

920.17

217.93

4

270

944.38

678.02

266.36

8

270

1113.89

944.38

169.50

Figure 2: Exercise 3, Numerical Data for Tyrosinase Activity

[DOPA] in mM

rate (umol/min) 1/S

1/v

0.5

76103.994

2

0.00001313

1

55880.555

1

0.00001789

2

48429.814

0.5

0.00002064

4

59191.995

0.25

0.00001689

8

37667.633

0.125

0.00002654

Figure 3: Michaelis Menten plot of Tyrosinase Activity **********sarah

The graph shows that as DOPA concentration increases, the initial rate of the reaction also increases. This is because, at a higher concentration of substrate, the enzyme is able to work at a higher velocity (until it reaches the Vmax when saturated with substrate). Figure 4: Exercise 4, Absorbance measured at 475 nm in varying concentrations of DOPA over a 5-minute reaction period WITH INHIBITOR Time (min)

1

2

3

4

5

6 (blank)

0

0.053

0.412

0

0.098

0.134

0

0.5

0.058

0.41

0.002

0.098

0.131

0

1

0.06

0.411

0.003

0.099

0.131

0

1.5

0.065

0.412

0.004

0.1

0.133

0

2

0.069

0.413

0.05

0.102

0.134

0

2.5

0.071

0.415

0.007

0.103

0.135

0

3

0.074

0.416

0.008

0.104

0.136

0

3.5

0.075

0.417

0.01

0.105

0.137

0

4

0.077

0.418

0.011

0.107

0.139

0

4.5

0.079

0.419

0.013

0.108

0.14

0

5

0.08

0.42

0.015

0.109

0.142

0

Figure: Exercise 4, concentrations

Tube

Tmax (min)

Conc max

1

5

1937.192584

1283.390087

653.802497

2

5

10170.26107

9976.541809

193.719261

3

5

363.2236095

0

363.2236095

4

5

2639.424896

2373.060916

266.36398

5

5

3438.516837

3244.797579

193.719258

6

5

0

Conc initial

Delta Conc

0

0

Figure 5: Numerical data for Tyrosinase activity in the presence of an inhibitor.

Tube

Rate (umol/min) 1/S

1/v

conc

1

130760.4994

0.1851851852

0.00000764756 5.4

2

38743.85168

0.25

0.00002581054 4

3

72644.72191

0.3703703704

0.00001376562 2.7

4

53272.79607

0.7692307692

0.00001877130 1.3

5

38743.85168

2

0.00002581054 0.5

6

0

0

0

0

Figure 6: Michaelis Menten of Tyrosinase activity in the presence of inhibitor **** sarah

Figure 7: Lineweaver Burke Plot comparing Tyrosinase activity in the absence and presence of inhibitor. *****************sarah

What were controls? What was unknown/test samples? Discussion and Conclusion: 40% **** The data obtained in lab is rather inconclusive. We expected to observe Michaelis Menten kinetics. While figure **** resembles a Michaelis Menten curve to an extent, it is not exactly the typical enzyme activity curve. Overall, though, the data shows that the change in absorbance increases as the concentration of DOPA increased. According to our Lineweaver Burke plots, we observed a change in *** and *** when comparing the uninhibited and inhibited conditions. This supports the idea that benzoic acid works as a *******, agreeing with my hypothesis. This is in agreement with prior research. Potential errors include inaccurate and imprecise measurements, failure to homogenize solutions prior to introducing subsequent serial dilutions or reactions, cloudy cuvettes, improper time keeping, and contamination in our solutions. Another large area for error is the fact that our

tyrosinase was isolated rather crudely. The extract could have contained proteases and other contaminants, not giving us a pure enzyme isolate.

References Körner, A., & Pawelek, J. (1982). Mammalian Tyrosinase Catalyzes Three Reactions in the Biosynthesis of Melanin. Science, 217(4565), 1163-1165. Retrieved from http://www.jstor.org/stable/1689654 Zhen Yang and Fengyin Wu, 2006. Catalytic Properties of Tyrosinase from Potato and Edible Fungi. Biotechnology, 5: 344-348. Retrieved from http://scialert.net/fulltext/?doi=biotech.2006.344.348 Gheibi, N., Taherkhani, N., Ahmadi, A., Haghbeen, K., & Ilghari, D. (2015). Characterization of inhibitory effects of the potential therapeutic inhibitors, benzoic acid and pyridine derivatives, on the monophenolase and diphenolase activities of tyrosinase. Iranian Journal of Basic Medical Sciences, 18(2), 122–129. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4366722/

Zhang, Karen J. "THE RATE OF TYROSINASE REACTION AND ITS ACTIVITY IN POTATO AND BANANA." IN POTATO AND BANANA (n.d.): n. pag. Web. 18 Oct. 2017.

Loizzo, M. R., et al. “Natural and Synthetic Tyrosinase Inhibitors as Antibrowning Agents: An Update.” Comprehensive Reviews in Food Science and Food Safety , Blackwell Publishing Inc, 12 June 2012, onlinelibrary.wiley.com/doi/10.1111/j.1541-4337.2012.00191.x/abstract....