Lab 4- Title Tyrosinase Enzyme Kinetics-2 PDF

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Lab 4 - full lab report...

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I. II.

Title: Tyrosinase Enzyme Kinetics Date: 02/04/19

III.

Lab Partners: Kimia Rezaeizadeh Tarlan Keshavarz

IV.

Purpose: The main purpose of this experiment was to observe enzyme activity under various conditions and measure the kinetics of enzyme catalysis with tyrosinase and its substrate DOPA. This lab further exposes the students to observe and quantitatively measure the effects of changes in enzyme concentration and substrate concentration on enzyme kinetics.

V.

Method:

Part 1: Preparation of a standard curve of the product

1. Obtain a ~6 mL aliquot of dopachrome from the front bench. 2. Label 7 plastic cuvettes using a Sharpie marker (#1-7). 3. Label the tyrosinase enzyme with your group's initials and invert the bottle to mix. 4. Use plastic cuvettes to prepare a series of dilutions as shown in the table below using a P200 and a P1000 pipetman 5. Use a P1000 to pipette up and down to mix. Do not cross-contaminate samples. 6. Refer to Appendix A for directions on using the Beckman DU 730 and Hach DR3900 spectrophotometers. 7. Read the absorbance of each cuvette at 475 nm • 8. Results from this data table will be used in the next sections. The reaction catalyzed by tyrosinase converts DOPA (substrate) dopachrome (product).

Part 2: Effects of enzyme concentration

1. Clean out two of your plastic cuvettes by pouring the dopachrome into the designated waste bottle, then rinsing each tube with DI water. 2. Invert the bottle of tyrosinase enzyme to

mix. 3. In a glass test tube, prepare 5 mL of a 1:5 dilution by mixing 1 mL tyrosinase enzyme + 4 mL citrate buffer. 4. Prepare the blank. In a plastic cuvette, prepare the blank by mixing: 1.6 mL of citrate buffer and 0.4 mL of diluted enzyme extract 5. In a second cuvette, place 1.6 ml of 5 mM DOPA (the substrate). 6. Use the Fixed Wavelength program on the spectrophotometer set to 475 nm. 7. Read the absorbance again at each timepoint. 8. Record the absorbance at each interval in a table similar to the one below. 9. If the absorbance exceeds 1.0 in less than 10 minutes, stop the experiment, dilute the enzyme extract further with citrate buffer and start again. 10. Depending on the absorbance readings make a second enzyme dilution either more/less dilute. 11. If the absorbance was less than 1.0 at 15 minutes, choose a more concentrated dilution if the absorbance was 1.0 or higher at 15 minutes, choose a less concentrated dilution 12. Repeat step 2 with the revised dilution and fill in the table below 13. Repeat for a third time if needed and add your results to the table below 14. Be sure the bottle of tyrosinase is labeled with your group's name. 15. Turn off and unplug your spec. Part 3: Effect of substrate concentration – Determination of KM and Vmax:

1. State the hypothesis for Part 3. 2. Determine the velocity of reaction by 2 µmoles/time 3. Plot velocity versus DOPA concentration 4. From the graph of V vs. [S], estimate the Vmax and KM values for tyrosinase. 5. Make a second graph of the data using the Lineweaver-Burk plot of 1/V vs 1/[S]. 6. Use the Lineweaver-Burk plot to calculate the Vmax and KM values.

7. The relationship between the Lineweaver-Burk plot compare and values obtained from the V. Part 4 – Substrate Specificity of Tyrosinase

1. Use the Fixed Wavelength setting on the Beckman spectrophotometer/Single wavelength setting on the Hach spectrophotometer. 2. Prepare a blank using 1.6 ml citrate buffer + 0.4 ml diluted enzyme 3. Prepare 4 cuvettes (A-D) following the table below, cover with parafilm and mix by inverting 3x. 4. Incubate on your benchtop for 10 min. 5. Read the absorbance at 475 nm and record results

Part 5 – Enzyme Inhibition

1. Prepare 2 cuvettes according to the table below, add 0.4 mL diluted enzyme to each cuvette and mix well.

Part 6 – Effect of Temperature

1. Prepare 5 cuvettes with 1.6 mL pyrocatechol solution. 2. After 5 minutes of substrate equilibration at various temperatures, add 0.4 mL diluted enzyme, mix & return to the designated temperature and incubate for 10 more minutes. 3. Removing 1 cuvette at a time, record the absorbance at 475 nm at the end of the 10 minute incubation.

Part 7 – Chemicals that modify enzymes

1. Prepare 3 test cuvettes (A-C) according to the table below, then add 0.4 mL diluted enzyme, mix thoroughly and incubate at the designated temperature for 10 minutes. Substrate + Chemical + Enzyme → Incubate 2. Record the absorbance at 475 nm. 3. Use the same blank as in Part 5.

VI.

Results: Part 1. Table 1. Table of absorbance values recorded after different molarities of Dopachrome were tested in spectrophotometer to obtain a standard curve. Tube/Cuvette

μL Dopachrome

μL Citrate Buffer

Amount of Dopachrome

Absorbance at 475 nm

1 (Blank)

0

1500

0 μmoles

0

2

100

1400

0.5 μmoles

0.010

3

150

1350

0.75 μmoles

0.012

4

200

1300

1.0 μmoles

0.022

5

400

1100

2.0 μmoles

0.040

6

800

700

4.0 μmoles

0.083

7

1200

300

6.0 μmoles

0.117

Figure 1. Standard curve of absorbance values against amount of Dopachrome in µmoles at 475 nm.

Part 2: Table 2. Absorbance values recorded at various time intervals. Two dilutions were tested, and both dilution recordings are shown below. First Analysis Enzyme diluted 1:5

Second Analysis Enzyme diluted 1:1

Time (min.)

Absorbance (475 nm)

Amount of Absorbance Dopachrome (475 nm) formed (μmol)

Amount of Dopachrome formed (μmol)

0

0.006

0.292

0.019

0.945

3

0.014

0.693

0.079

3.96

5

0.026

1.30

0.124

6.22

10

0.033

1.65

0.151

7.58

15

0.045

2.25

0.178

8.94

Figure 2. Scatter plot comparing the effects of enzyme concentration on the amount of Dopachrome formed in µmoles.

Part 3: Hypothesis: An increased substrate concentration will increase the rate of the reaction until the enzyme is saturated. Table 3.1. Table displaying different dilutions of enzyme Tyrosinase to obtain the optimal concentration. Dilution Tested 1:1

Time to desired absorbance (min)

Abs at 3 min

3.33

0.039

Abs. at 5 min 0.039

Table 3.2. Table displaying preparations of cuvettes tested for enzyme activity with different substrate concentration. DOPA concentrati on (mM)

Vol DOPA (mL)

Vol diluted enzyme (mL)

Time Abs (475 required to nm) form 2 μmoles of Dopachrom e (min.)

Rate of DOPA Conversion (V) (μmoles Dopachrom e /min) = [product]/ time

10

1.6 mL

0.4 mL

3.53

0.567

20

1.6 mL

0.4 mL

2.41

0.830

25

1.6 mL

0.4 mL

1.30

1.54

30

1.6 mL

0.4 mL

0.67

2.99

Figure 3.1. Graph displaying the effect of the DOPA substrate concentration on the rate of the DOPA conversion. Calculations: ½ Vmax = Km: Vmax= ~3.2 ½ Vmax= 1.6, y = 0.01x2 − 0.2795x + 2.3749 1.6 = 0.01x2 − 0.2795x + 2.3749 K M = 25.4 mM

Figure 3.2. Lineweaver-Burke plot displaying the inverse of the graph in Figure 3.1. in order to obtain a linear graph and equation. Calculations: x-intercept = -1/Km x-intercept= -0.0368 Km= 27.2 y-intercept= 1/Vmax y-intercept= 0.4577 Vmax= 2.18

7. The values from the Lineweaver-burke plot are more accurate because they come directly from the equation of the line. The values from the V vs. [S] plot are estimated, but overall the values are relatively similar. Part 4: Table 4. Displaying preparations of cuvettes tested for enzyme activity with a variety of substrates. Cuvette

Substrate (mL)

Diluted Enzyme (mL)

Abs (475) @ 10 min

Blank

Citrate Buffer, 1.6mL

0.4

NA

A

5 mM DOPA, 1.6 mL

0.4

Pyrocatechol, 1.6 mL

0.4

B C

Hydroquinone, 1.6 mL

0.050 0.001 0.4

-0.043=0

Table 4. Bar graph comparing enzyme activity of Tyrosinase with varying substrates. The substrate that resulted in the highest activity was 5mM DOPA.

Part 5: Table 5. Displaying preparations of cuvettes tested for enzyme activity with different inhibitors present. Cuvette

Substrate (mL)

Diluted Enzyme

Inhibitor (mL) Abs (475) @ 10 min

Blank

Citrate Buffer, 0.64 mL

0.4 mL

dH2O, 0.96 mL

NA

A

Pyrocatechol, 0.64 mL

0.4 mL

dH2O, 0.96 mL

0.026

B

Pyrocatechol, 0.64 mL

0.4 mL

Benzoic acid, 0.96 mL

0.007

Figure 5. Bar graph comparing enzyme activity of Tyrosinase using absorbance readings with different inhibitors present. Water and Benzoic acid were used to inhibit the enzyme.

Part 6: Table 6. Table displaying preparations of cuvettes tested for enzyme activity in varying temperatures. Cuvette

Temperature

Substrate

Diluted enzyme** add after 5' equilibration

Abs (475) @ 10 min

Blank

22 (room temp.)

1.6mL pyrocatechol

0.4 mL citrate buffer

N/A

A

0

0.033

B

0.4 mL tyrosinase

22 (room temp.)

0.059

C

37 (water bath)

0.041

D

70 (water bath)

0.044

(on ice)

Figure 6. Bar graph comparing enzyme activity of Tyrosinase using absorbance readings under different temperature conditions. Highest activity correlates with highest absorbance at 22°C.

Part 7: Table 7. Table displaying preparations of cuvettes tested for enzyme activity with varying chemicals present. Tube

Substrate (ml)

Diluted enzyme

Chemical (ml)

Temp. (°C)

Abs @ 10 min

Blank

Pyrocatech ol, 0.64 ml

0.4 mL tyrosinase

dH2O, 0.96 ml

37

NA

A

Pyrocatech ol, 0.64 ml

Pepsin, 0.96 37 ml

0.029

B

Pyrocatech ol, 0.64 ml

TCA*, 0.96 ml

-0.016=0

room temp.

Figure 7. Bar graph comparing activity of Tyrosinase with chemicals Pepsin and trichloroacetic acid.

VII.

Discussion

1. What is the relationship between tyrosinase, tyrosine, and melanin? Hint: look up the structures of L-DOPA & melanin online. Tyrosinase is a biological enzyme that reacts with the substrate, tyrosine, to create a pigment called melanin. Melanin is the pigment that allows for eyes, hair, and skin color. Firstly, Tyrosinase converts the amino acid tyrosine to dopaquinone. This dopaquinone can be converted to L-DOPA, which is eventually converted into melanin through a series of other reactions.

2. Did the plot of V vs. [S] (from question #2 in data analysis above) confirm your previous hypothesis of how substrate concentration would affect the rate of reaction? Why or why not? The plot of Velocity (V) vs. substrate concentration [S] confirms the hypothesis that an increased substrate concentration will increase the rate of the reaction. More substrate in solution will allow the enzyme to catalyze the reaction, therefore producing more product. An increased product will result in a higher absorption value due to the color formation. However, increasing the enzyme concentration increases the maximum reaction rate, and enzymes become saturated when the substrate concentration is high and hits a plateau.

3. Consult your textbook to obtain KM values for several other enzymes (check units!). List these enzymes and their KM values in a table. Based on your estimate of the KM for tyrosinase and compared to these other known enzymes, does tyrosinase require a large or small concentration of substrate to yield product? (Remember that a low KM value indicates the reaction is at 1⁄2 of Vmax at a low concentration of substrate.) The Km for tyrosinase was experimentally determined to be 27.2 mM. Tyrosine Km is higher to compare with the values in the table below. We can conclude that Tyrosine is less sensitive to the substrate and it needs a higher concentration to yield product to reach Vmax. Substrate

Enzyme

Km (mM)

Acetyl-L-tryptophanamid e

Chymotrypsin

5.00

Hexa-N-acetylglucosamin e

Lysozyme

0.006

Lactose

β- Galactosidase

4.00

CO2

Carbonic Enzyme

8.00

Benzylpenicillin

Penicillinase

0.050

4. What are some sources of experimental error in this experiment? How might these errors be minimized? The source of experimental errors in this experiment are, Km and Vmax values of inhibitors are nearly the same or uncompetitive. If the solution is not kept sufficiently cold throughout the experiment. Errors in UV-vis spectrophotometer readings that generally appear when the timing of tyrosinase addition varies a lot for different test tubes.

5. Which substrate(s) does tyrosinase act upon? Based upon comparisons of the structures, are these results expected? Why or why not? Tyrosinase is an enzyme that oxidizes monophenols and diphenols. In this lab, the substrates DOPA, pyrocatechol and hydroquinone were used. The L-DOPA was the most effective, which is expected as it is the ideal substrate for Tyrosinase. Its structure is a phenol with an additional carboxyl group attached. Pyrocatechol has a similar structure to DOPA, which is why it is an alternate substrate for Tyrosinase as the hydroxyl groups are attached at adjacent carbon. Hydroquinone is also an alternate substrate for Tyrosinase and is an structural isomer of pyrocatechol. Pyrocatechol and hydroquinone differ in their redox properties as well as the placement of their hydroxyl groups. The hydroquinone showed less activity with Tyrosinase at 475 nm providing a lower absorption of zero.

Pyrocatechol

Hydroquinone

L-DOPA

6. Look up the structure of benzoic acid. How does the presence of benzoic acid affect the activity of tyrosinase and pyrocatechol? Based on your comparisons of the structures of pyrocatechol and benzoic acid, might you expect benzoic acid to be a competitive inhibitor? (Note that benzoic acid is a weak acid and will not damage the enzyme through direct chemical action.) How could you experimentally determine whether or not benzoic acid is a competitive inhibitor? The inhibition kinetics and mechanism of the enzyme show that the inhibition of tyrosinase by benzoic acid is a reversible reaction with remaining enzyme activity. The combining of benzoic acid with the enzyme molecule resulted in decreasing of the quantal rate of the enzyme intrinsic fluorescence without apparent position-shifted. Both benzoic acid and pyrocatechol have aromatic ring which is the section that will bind to the active site. The inhibitor is similar in structure to the substrate. To experimentally test the benzoic acid, a solution of Tyrosinase and pyrocatechol could be tested against a solution of Tyrosinase, pyrocatechol, and benzoic acid. If the second solution shows a lower absorbance value, then it will indicate that the Tyrosinase is unable to act upon the pyrocatechol with the presence of benzoic acid since it’s a competitive inhibitor.

7. At what temperatures is tyrosinase active? At what temperatures is it inactive? Give a molecular explanation for the effects of temperature on tyrosinase activity, particularly 0 and 70o C (look online). If you did not get the expected result, explain what may have happened. Tyrosinase temperature-sensitive the enzyme is inactive in cells grown at 37 degrees C but displays full activity in cells grown at 31 degrees C. Tyrosinase is maximally active at temperatures well below normal body temperature. (15 degrees C to 25 degrees C). This was also proven experimentally, where Tyrosinase was most active at 22°C, with the highest absorption reading (0.059), implying that the most activity occurred at this temperature. Our experimental results were expected and as they reflect the thermodynamics behind the enzyme activity and temperature as seen in the graph below. Enzyme activity increases with an increased temperature up to approximately 37°C (warm water bath), then the heat begins to denature the enzyme at a higher temperature. The expected results would be a low activity in the enzyme kept at 0°C and in the hot water bath, then slightly increased activity at room temperature, with a final optimal temperature of 37°C.

8. How do pepsin and trichloroacetic acid affect the activity of tyrosinase? (Consult your textbook or other references for information on these compounds and their effects on proteins.) The TYR gene provides instructions for making an enzyme called Tyrosinase. This enzyme is located in melanocytes, which are specialized cells that produce a pigment called melanin. Melanin is the substance that gives skin hair and eyes their color. Pepsin is an enzyme produced in the mucosal lining of the stomach that acts to degrade protein.It is an enzyme that break down proteins into smaller peptides. Pepsin is one of the principal proteolytic enzymes in the digestive system. Pepsinogen is converted into pepsin. Trichloroacetic acid is widely used in the biochemistry for the precipitation of macromolecules such as protein, DNA, RNA. When it applied to skin top layers of skin dry up and peel off over a period of several days. The pepsin and trichloroacetic acid act as inhibitors of tyrosinase activity and melanin pigmentation. The pepsin may also help to inhibit the activity of tyrosinase, consequently helping the inhibition of melanin pigmentation. In the same way, trichloroacetic acid used in the precipitation of macromolecules, they can affect the tyrosinase enzyme activity....