Exercise 5.

1 - Extraction of Tyrosinase
LEVEL I
Materials

Potatoes
Paring Knife
Blender
0.1 M NaF
Rubber Gloves
Saturated Ammonium Sulfate (4.1 M @25 C)
Volumetric Cylinders (50 ml, 100 ml, 250 ml)
Cheesecloth
Beakers (100 ml, 250 ml)
Chilled Centrifuge Tubes (30-50 ml)
Refrigerated Centrifuge
0.1 M Citrate Buffer, pH 4.8
Glass Stirring Rod

Tyrosinase also
known
as monophenol
monooxygenase is
an enzyme that catalyses the oxidation of phenols (such astyrosine) and is widespread
in plants and animals. Tyrosinase is a copper-containing enzyme present in plant and animal
tissues that catalyzes the production of melanin and other pigments from tyrosine by oxidation, as
in the blackening of a peeled or slicedpotato exposed to air. It is found inside melanosomes. In
humans, the tyrosinase enzyme is encoded by the TYR gene.[1]

Catalyzed reaction
Tyrosinase carries out the oxidation of phenols such
as tyrosine and dopamine using dioxygen (O2). In the presence
of catechol,benzoquinone is formed (see reaction below). Hydrogens removed from
catechol combine with oxygen to form water.

Homogenization (cell biology) is a process that involves breaking apart cells releasing
organelles and cytoplasm.

When the purpose is to extract organelles, it is frequently done in two steps; first using
a blender to break the tissue up, and then with an ultrasonic or mechanical tissue disruptor. The
organelles are then generally separated using differential centrifugation.

Procedure
1. Peel a small potato and cut into pieces about 1 inch square.
2. Add 100 grams of the potato to a blender, along with 100 ml of sodium fluoride
(NaF). Homogenize for about one minute at high speed.
Caution: Sodium Fluoride is a poison! Wear rubber gloves while handling,
and wipe up any spills immediately.
3. Pour the homogenate (mixture) through several layers of cheesecloth and into a
beaker.
4. Measure the volume of the homogenate and add an equal volume of saturated
ammonium sulfate. That is, if the fluid volume of your homogenate is 150 ml, add
150 ml of ammonium sulfate. This will cause a flocculent white precipitate to
appear as many of the previously soluble potato proteins become insoluble. The
enzyme tyrosinase is one of these proteins and thus will be found in the
subsequent precipitate.
5. Divide the ammonium sulfate-treated homogenate into chilled centrifuge tubes
and centrifuge at 1,500 xg for 5 minutes @ 4 C.
6. Collect the centrifuge tubes, and carefully pour off and discard the fluid
(supernatant). Save the pellets. Combine all of the pellets into a 100 ml beaker.
7. Add 60 ml. of citrate buffer, pH 4.8, to the pooled pellet and stir the contents well.
Use a glass rod to break up the pellet. Continue to stir for 2 minutes while keeping
the solution cool.
8. Again divide the solution into centrifuge tubes and re-centrifuge at 300 xg for 5
minutes at 4 C.
9. Collect and save the supernatant. This is your enzyme extract! Place it in an
erlenmeyer flask, label it as Enzyme Extract and place it in an ice bucket. The
enzyme tyrosinase is insoluble in 50% ammonium sulfate, but is soluble in the
citrate buffer. Keep this extract chilled for the duration of the laboratory.
Tyrosinase under is stable for about an hour the conditions of this exercise. If not
used within this period, you will need to extract more enzyme from a fresh potato.

Exercise 5.2 - Preparation of Standard

Curve
LEVEL I

Materials

8 mM DOPA
Enzyme Extract - From Exercise 5.1 (about 60 ml)
Test tubes
5 ml Pipette
0.1M Citrate Buffer, pH 4.8
Spectrophotometer and Cuvettes

Procedure
1. Begin by preparing a standard solution of the orange colored dopachrome from LDOPA [L-3,4 DIHYDROXYPHENYLALANINE]. To 10 ml of 8 mM DOPA,
add 0.5 ml of your enzyme extract 4 and allow the solution to sit for 15 minutes at
room temperature. During this period, all of the DOPA will be converted to
dopachrome, and your solution will now contain 8 mM dopachrome. Dopachrome
is somewhat unstable in the presence of light and should be stored in an amber
bottle or out of the light.
2. Prepare a 1:1 series of dilutions of the 8 mM Dopachrome to yield the
concentrations in the following table: Add 3.0 ml of each indicated concentration
to tubes #1-8.
Tube #

Final Concentration
of Dopachrome (mM)

0.125

0.25

0.5

1.0

2.0

4.0

8.0

3. With these dilutions, you have prepared tubes containing concentrations

from 0 to 8 mM dopachrome (tubes 1-8). Tube 1 contains no dopachrome
and is used for blanking the spectrophotometer.
4. The units of concentration are millimolar (mM). A 1.0 mM solution
contains .001 moles per liter or .000001 moles per ml. Thus, with a volume
of 3.0 ml, there are .000003 moles of dopachrome, or 3 micromoles.
Correspondingly, tubes 2-8 contain 1 to 24 micromoles of dopachrome. For

the remainder of this exercise, be sure to distinguish between concentration

(mM) and total amount of substance present (micromoles).
5. Turn on your spectrophotometer and set a wavelength of 475. Use tube #1 from
the above dilutions as a blank and adjust the spectrophotometer for 0 and 100% T.
Read the absorbance (or read and convert transmittance) of each of the solutions
in tubes 2-8 and complete the following table:
[add BEER-LAMBERT LAW THEORY]
Tube

Concentration
of
Absorbance A/C
Dopachrome (mM)

#1

#2

0.125

#3

0.25

#4

0.5

#5

1.0

#6

2.0

#7

4.5

#8

8.0

----

6. Calculate the values for the last column of the table. This column represents the
simplest calculation of the extinction coefficient for dopachrome absorbance.[]
Average the values in this column and enter the number at the bottom of the
column. This is the average extinction coefficient and can be used in subsequent
determinations of dopachrome concentrations according to the Beer-Lambert law.
the molar extinction coefficient are parameters defining how strongly a substance
absorbs light at a given wavelength, per mass density or per molar concentration,
respectively.
The molar
absorption
coefficient, molar extinction
coefficient,
or molar
absorptivity, is a measurement of how strongly a chemical species absorbs light at a
givenwavelength. It is an intrinsic property of the species; the actual absorbance, A, of a
sample is dependent on the pathlength, , and the concentration, c, of the species via
theBeerLambert law,
.
The SI units for are m2/mol, but in practice, they are usually taken as M1 cm1 or L
mol1 cm1. In older literature, cm2 mol1 is sometimes used with corresponding values
1000 times larger. These units may look different, but it is just a matter of expressing
volume in cm3 or in L.
the extinction coefficient of a protein at 280 nm depends almost exclusively on the
number of aromatic residues, particularly tryptophan, and can be predicted from the
sequence of amino acids.[2] If the extinction coefficient is known, it can be used to
determine the concentration of a protein in solution.

Prerequisites
There are at least six conditions that need to be fulfilled in order for Beers law to be valid. These
are:
1. The absorbers must act independently of each other;
2. The absorbing medium must be homogeneous in the interaction volume
3. The absorbing medium must not scatter the radiation no turbidity;
4. The incident radiation must consist of parallel rays, each traversing the same length in
the absorbing medium;
5. The incident radiation should preferably be monochromatic, or have at least a width
that is narrower than that of the absorbing transition; and
6. The incident flux must not influence the atoms or molecules; it should only act as a noninvasive probe of the species under study. In particular, this implies that the light should
not cause optical saturation or optical pumping, since such effects will deplete the lower
level and possibly give rise to stimulated emission.
If any of these conditions are not fulfilled, there will be deviations from Beers law.

Limitations of the Beer-Lambert law

The linearity of the Beer-Lambert law is limited by chemical and
instrumental factors. Causes of nonlinearity include:

deviations in absorptivity coefficients at high concentrations (>0.01M) due

to electrostatic interactions between molecules in close proximity
scattering of light due to particulates in the sample
fluoresecence or phosphorescence of the sample
changes in refractive index at high analyte concentration
shifts in chemical equilibria as a function of concentration
non-monochromatic radiation, deviations can be minimized by using a
relatively flat part of the absorption spectrum such as the maximum of an
absorption band
stray light

You can more accurately determine the extinction coefficient by performing

a linear regression analysis of your data, and computing the slope and y
intercept. The slope of the linear regression will represent the extinction
coefficient for your sample.
7. Plot a scattergram of the absorbance value against the concentration of
dopachrome. The known concentration of dopachrome should be the x axis, while
absorbance should be the y axis.
8. Plot the computed slope and intercept of the linear regression as a straight line
overlaying your scattergram. The equation for a straight line is y = mx + b, where
m is the slope and b the intercept. 5

Notes
Since tyrosinase catalyzes the conversion of L-DOPA to dopachrome, this exercise
measures the conversion of colorless DOPA to the dark orange dopachrome.
Substrate and product are in a 1:1 ratio for this reaction, thus the amount of
product formed equals the amount of substrate used. The optical density of

dopachrome @475 nm is directly proportional to the intensity of orange color

formation in solution (Beer-Lambert Law).

Exercise 5.3 - Enzyme Concentration

LEVEL I
Materials

Enzyme Extract
0.1 M Citrate Buffer, pH 4.8
10 ml Pipette
8 mM DOPA
Spectrophotometer and Cuvettes
Ice Bath

Procedure
1. To determine the kinetic effects of the enzyme reaction, first determine an
appropriate dilution of your enzyme extract. This will give a rate of reaction of 510 micromoles of DOPA converted per minute. Prepare a serial dilution of your
enzyme extract. Place 9.0 ml of citrate buffer into each of three test-tubes. Label
the tubes 1/10, 1/100 and 1/1000.
2. Pipette 1.0 ml of your enzyme extract into the first of these tubes (the one labeled
1/10) and mix by inversion.
3. Pipette 1.0 ml of the 1/10 dilution into the second tube (labeled as 1/100) and mix
by inversion.
4. Pipette 1.0 ml of the 1/100 dilution into the third tube (labeled as 1/1000) and mix
by inversion.
5. Place all of the dilutions in the ice bath until ready to use.
6. If not already done, turn on a spectrophotometer, adjust to 475 nm and blank with
a tube containing 2.5 ml of citrate buffer and 0.5 ml of enzyme extract.
7. Add 2.5 ml of 8 mM DOPA to each of 4 cuvettes or test tubes. Note that each tube
contains .0025 X .008 moles or 20 micromoles of DOPA.
8. Add 0.5 ml of undiluted enzyme extract to one of the tubes containing the 8 mM
DOPA. Mix by inversion, place into the spectrophotometer and immediately
begin timing the reaction. Carefully measure the time required for the conversion
of 8 micromoles of DOPA. Note that since the cuvette will contain a volume of
3.0 ml, the concentration when 8 micromoles are converted will be 8/3.0 or 2.67
mM dopachrome. Use the data from the standard curve (Exercise 5.2) to
determine the absorbance equal to 2.67 mM dopachrome. This absorbance value
will be the end point for the reaction.
The Absorbance equal to 3.33 mM dopachrome (from Exercise 5.2) = _______

9. As the reaction takes place within the spectrophotometer, the absorbance

will increase as dopachrome is formed. When the absorbance reaches the
value above, note the elapsed time from the mixing of the enzyme extract
with the 10 mM DOPA. Express the time as a decimal rather than minutes,
seconds. The time should be between three and five minutes. If the end
point is reached before three minutes, repeat Step 8, but using the next
dilution of enzyme (i.e. the 1/10 after the undiluted, the 1/100 after the 1/10
and the 1/1000 after the 1/100).
The rate of activity = ________________ micromoles/minute/0.5 ml of diluted
extract.

The dilution factor (inverse of dilution, 1,10,100 or 1000) is ______.

The
activity
of
the
micromoles/minute/0.5 ml

undiluted

enzyme

is

____________

or _____________ micromoles/minute/1.0 ml of extract.S

10. For the enzyme dilution which reaches the end point between 3 and 5 minutes,
calculate the velocity of reaction. Divide the amount of product formed (10
micromoles) by the time required to reach the end point.

Exercise 5.4 - Effects of pH

LEVEL I
Materials

8
mM
DOPA
in
citrate
pH values of 3.6, 4.2, 4.8, 5.4, 6.0, 6.6, 7.2, 7.8
Enzyme Extract
Spectrophotometer and Cuvettes
Stopwatch

buffer

adjusted

to

Procedure
1. Set up a series of test tubes each containing 2.5 ml of 8 mM DOPA, but adjusted
to the following pH values: 3.6, 4.2, 4.8, 5.4, 6.0, 6.6, 7.2, and 7.8
2. Begin with the tube containing DOPA @ pH 3.6, add 0.5 ml of the diluted enzyme
extract which will convert 10 micromoles of DOPA in 3-5 minutes as determined

in Exercise 5.3. Start timing the reaction, mix by inversion and insert into the
spectrophotometer. Note the time for conversion of 10 micromoles of DOPA.
3. Repeat Step 2 for each of the indicated pH values. Complete the following table:
pH

Time(Minutes)

Micromoles of
Dopachrome

3.6

10

4.2

10

4.8

10

5.4

10

6.0

10

6.6

10

7.2

10

7.8

10

Velocity
(Micromoles/Minute)

4. Plot pH (x-axis) versus Reaction Velocity (y-axis)

Exercise 5.5 - Effects of Temperature

LEVEL I
Materials

Enzyme Extract
8 mM DOPA pH 6.6
Incubators or Water baths adjusted to 10, 15, 20, 25, 30, 35 and 40 C 6
Spectrophotometer and Cuvettes
Stopwatch

Procedure
1. Set up a series of test tubes each containing 2.5 ml of 8 mM DOPA buffered to a
pH = 6.6. Place one tube in an ice bath or incubator adjusted to the following
temperature; 10, 15, 20, 25, 30, 35 and 40 C

2. Add 0.5 ml of an appropriately diluted enzyme extract (to yield 10 micromoles

dopachrome in 3-5 minutes) to each of a second series of tubes. Place one each in
the corresponding temperature baths. Allow all of the tubes to temperature
equilibrate for 5 minutes. Do not mix the tubes.
3. Beginning with the 10 C tube, and with the spectrophotometer adjusted to 475
nm and properly blanked, pour the enzyme (0.5 ml @ 10 C ) into the tube
containing the DOPA and begin timing the reaction. Mix thoroughly. Note the
time to reach the end point equivalent to the conversion of 10 micromoles of
substrate.
4. Repeat Step 3 for each of the listed temperatures, complete the following and plot
the data.
Temperature ( C )

Time
Micromoles of Velocity
(Minutes) Dopachrome (Micromoles/Minute)

10

10

15

10

20

10

25

10

30

10

35

10

40

10

Exercise 5.7 - Kinetic analysis

LEVEL II
Materials

8 mM DOPA pH 6.6
Enzyme Extract, diluted to yield 10 micomoles of dopachrome in 3-5 minutes
(Exercise 5.2)
Spectrophotometer and Cuvettes
Stopwatch

Procedure
1. Prepare a reaction blank in a clean cuvette to contain 2.5 ml of citrate buffer and
0.5 ml of enzyme extract. Use this blank to adjust your spectrophotometer for
100% transmittance. Remove the blank and save.

2. Add 2.5 ml of 8 mM DOPA, pH 6.6 to a clean cuvette.

3. Add 0.5 ml of appropriately diluted enzyme extract. Shake well and immediately
insert the tube into the spectrophotometer. Record the absorbance or transmittance
as quickly as possible. Designate this reading as time 0.
4. At 30 second intervals read and record the transmittance until a transmittance
value of 10% (Absorbance = 1.0) is reached. Complete the following table:
Time
Concentration of Micromoles of
Absorbance
(Minutes)
Dopachrome(mM) Dopachrome
0.5
1.0
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5. Plot time in minutes (x-axis) versus the amount of dopachrome formed (y-axis).

Exercise 5.8 - Determination of Km and

Vmax
LEVEL II
Materials

Enzyme Extract
8 mM L-DOPA adjusted to pH 6.6
Spectrophotometer and Cuvettes
Stopwatch

Procedure
1. Dilute the DOPA standard (8 mM) to obtain each of the following concentrations
of L-DOPA: 0.5 mM, 1 mM, 2 mM 4 mM, and 8 mM.

2. Repeat Exercise 5.7 for each of the substrate concentrations listed, substituting the
change in concentration where appropriate.
3. Plot each set of data and from the data calculate the time required to convert 10
micromoles of DOPA to dopachrome. Compute the velocity of enzyme reaction
for each substrate concentration. Fill in the following table:9
Substrate
(DOPA)
Concentration (mM)

Velocity
Micromoles/Minute

1/s

0.5

2.00

1.0

1.00

2.0

0.50

4.0

0.25

8.0

0.125

1/v

4. Plot the rate of DOPA conversion (v) against substrate concentration in the
appropriate place below. This is a Michaelis-Menten plot.
5. _______________________________________________________________
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_______________________________________________________________|

Michaelis-Menten plot
22. Plot a double reciprocal of the values plotted in step 4; that is, 1/s versus 1/v. This
is a Lineweaver-Burke plot.
23.
24.
25.
26.
27.
28.

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29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.

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Lineweaver-Burke plot
40. Perform a linear regression analysis on the second plot and compute the slope and
both y and x intercepts.

Note that the x intercept is -1/K , the negative inverse of which is the MichaelisMenten Constant. The y intercept is 1/V and the slope equals Km/V .

Exercise 5.9 - Addition of Enzyme

Inhibitors
LEVEL II
Materials

Enzyme Extract
8 mM L-DOPA
8 mM Benzoic Acid
8 mM KCN
0.1 M Citrate Buffer, pH 6.6

Procedure
1. Tyrosinase is inhibited by compounds that complex with copper, as well as by
benzoic acid and cyanide. To determine the inhibitory effects of benzoic acid and
cyanide, set up a series of tubes as indicated.
2. Using one tube at a time, add 0.5 ml of the enzyme dilution previously calculated
to yield 10 micromoles of dopachrome in 2-3 minutes. For each tube, measure the
time required to convert 10 micromoles of DOPA to dopachrome. Enter those
times in the table below. Compute the reaction velocity for each substrate
concentration:
Tube #

Time

Final [DOPA] nM

Velocity

(Minutes)

Micromoles/Minute

1Benzoic Acid

6.67

2 Inhibited

6.00

3 Series

5.33

4.67

4.00

3.33

2.67

2.00

1.33

10

0.67

11

12 KCN

6.00

13 Inhibited

5.33

14 SERIES

4.67

15

4.00

16

3.33

17

2.67

18

2.00

19

1.33

20

0.67

21

3. Calculate the values for 1/s and 1/v for each of the corresponding s and v in the
table below. Plot 1/v vs 1/s for the presence of benzoic acid and a second plot for
the presence of KCN. Compute the values of V
and K for the presence of
each inhibitor. Determine whether these inhibitors are competitive, noncompetitive or uncompetitive.
Benzoic Acid Inhibition
Tube #

8 mM DOPA

8 mM Benzoic Acid

Buffer

2.0

0.5

1.8

0.5

0.2

1.6

0.5

0.4

1.4

0.5

0.6

1.2

0.5

0.8

1.0

0.5

1.0

0.8

0.5

1.2

0.6

0.5

1.4

0.4

0.5

1.6

10

0.2

0.5

1.8

11

0.5

2.0

KCN Inhibition

Tube #

8 mM DOPA

8 mM KCN

Buffer

12

1.8

0.5

0.2

13

1.6

0.5

0.4

14

1.4

0.5

0.6

15

1.2

0.5

0.8

16

1.0

0.5

1.0

17

0.8

0.5

1.2

18

0.6

0.5

1.4

19

0.4

0.5

1.6

20

0.2

0.5

1.8

21

0.5

2.0

Exercise
5.10
Protein
Concentration/Enzyme Activity
LEVEL II
Materials

Commercially pure tyrosinase

UV-Spectrophotometer
or Materials for Lowry or Bradford Protein determination
L-DOPA
0.1 M Citrate buffer, pH 6.6

Procedure
1. Prepare a solution of 0.7 micrograms of commercially pure tyrosinase diluted to 4
ml. with 0.1 M Citrate Buffer, pH 6.6.

2. Measure the OD of your sample and prepare a dilution of your enzyme extract
to a final concentration of 0.7 micrograms in 4 ml of citrate buffer.
3. Place both enzyme samples in a water bath @ 30 C for 5 minutes to temperature
equilibrate.
4. Turn on the spectrophotometer, set the wavelength to 475 nm and blank the
instrument using citrate buffer as the blank.
5. Select the commercial preparation and add exactly 1.0 ml of L-DOPA (4 mg/ml in
citrate buffer) and immediately read the absorbance at 475 nm.
6. Replace the tube in the water bath and wait exactly 5 minutes. Read the OD
immediately.
7. The molar absorbance coefficient for dopachrome is 3.7 x 10 . Use this value to
compute the specific activity of the commercial enzyme preparation. Check this
activity against that listed with the enzyme preparation.
8. Repeat steps 5 and 6 with your extracted enzyme preparation. Compute the
specific activity (enzyme units of activity/ mg protein) of your enzyme
preparation. Enzyme Unit: The absorbance reading under the conditions specified
in this exercise is proportional to the enzyme concentration, where 1 unit of
enzyme activity yield a 0.81 OD change in readings.

Notes
The protein content can be measured by the Lowry or Biuret procedures found in
the Appendix G, or more simply by a single spectrophotometric measure of the
absorbance of the sample at 280 nm. Without going into mathematical detail, a 1%
pure solution of tyrosinase has an OD equal to 15.6/cm. The Beer-Lambert law
can thus be used to determine protein content in a non-destructive manner.