BIOLS 315

Biochemistry Lab. No. 4

Enzyme Kinetics
Determination of Michaelis Menten Constant and Maximum Velocity of
Alkaline Phosphatase

Objectives

1. To determine the initial rate and the velocity of the reaction by varying substrate
concentration.
2. To study and verify the Michaelis Menten (MM) behavior of the alkaline
phosphatase.
3. To perform the double reciprocal (Lineweaver-Burk) plot.
4. To calculate the MM constant (KM) and the Maximum velocity (Vmax).

Alkaline Phosphatase Assay

Requirements

. Prepare a stock solution of 200 mM of p-nitrophenyl phosphate (p-NPP) and

store in a brown bottle & freeze.
. From this prepare the second stock solution of 20mM (50 ml), store in a
brown bottle and freeze until use.
. On the day of the lab, prepare 8 reaction mixtures according to the following
table (Final volume 1.0 ml each) in brown bottles.

Component Substrate mM

0.125 0.25 0.33 0.417 0.58 0.87 1.67 4.0

1 2 3 4 5 6 7 8
20mM p-NPP (μl) 125 250 333 417 580 870 - -
200 mM p-NPP (μl) - - - - - - 167 400
H2O (μl) 875 750 666 583 420 130 833 600
Final volume (ml) 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

Procedure

1. Lable two sets of 8 test tubes (13X 100 mm).

2. Add 0.9 ml of 0.1 M glycine-NaOH buffer into each test tube.
3. Dispense 50 μl of each reaction mixture into the corresponding test tube.
4. Add 50 μl enzyme solution, mix well.
5. Prepare a blank solution by mixing 0.95 ml of 0.1 M glycine-NaOH buffer and 50
μl of the enzyme solution.
6. Stop the reaction by adding 2.0 ml of 0.1 M NaOH to one set of test tubes
only.
7. Read the absorbance at 410nm for each tube.

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8. Incubate the second set at 37ºC for 10 min.
9. Stop the reaction by adding 2.0 ml of 0.1 M NaOH.
10. Measure the absorbance at 410 nm for each tube.
11. Calculate the number of moles (μ, n) of the product formed using
12. A= ε. l .c where ε =21,000M-1 cm-1

Component Reaction mixture

0.125 0.25 0.33 0.417 0.58 0.87 1.67 4.0

1 2 3 4 5 6 7 8
Glycine-NaOH (ml) 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9
Substrate p-NPP (μl) 50 50 50 50 50 50 50 50
Enzyme solution (μl) 50 50 50 50 50 50 50 50
Mix, incubate at 37oC for 10 min. Stop the reaction by adding
NaOH (ml) 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0

 Test tube number 0 (blank): 0.95 ml Glycine-NaOH + 50 μl Enzyme solution.

Mix, incubate at 37oC for 10 min. Stop the reaction by adding 2.0 ml of
NaOH.

Assignment
 Plot Michaelis-Mention and Lineweaver-Burk using the information from the
table below. Find out the KM and Vmax values from both plots.
 Compare KM and Vmax values obtained from Lineweaver-Burk to those
obtained from MM curve.

Table 1: The reaction velocity with different substrate concentration.

Substrate Absorbance Absorbance Velocity (v) p- 1/v
at 410nm at 410nm NPP produced
per
Tube [S] 1/[S] 0 min 10 min 10 min 1 min
#
1 0.125 0.002 0.110
2 0.25
3 0.33
4 0.417
5 0.58
6 0.87
7 1.67
8 4.0

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Example of calculation

Test tube 1
 Calculate the concentration of products
Absorbance at 10 min – absorbance at 0 min = 0.110 - 0.002 = 0.108
Calculate the number of moles (M) of the product formed using
A= ε. l .c where ε =21,000M-1 cm-1
A= *I*C
0.108=21000*1*C

C= = 5.14 x 10-6 M

 Convert to millimole/10 min: Divide by 10-3

5.14 x 10-6 /10-3 = 5.14 x 10-3 mM/10 min
Convert to millimole/1 min: Divide by 10
5.14 x 10-3 mM/10 min = 0.514 x 10-3 mM/1 min

 Repeat the calculation for test tubes 2-8