1. ASSAY OF Km VALUE OF ALKALINE PHOSPHATASE
EXPERIMENT 4
ASSAY OF Km VALUE OF ALKALINE PHOSPHATASE

2. Aims
1. To understand the effect of the changes of substrate concentrations on the velocity of enzyme-catalyzed reaction.
2. To learn the principle and methods of determining Km value and its role at enzyme kinetics.

3. velocity of enzyme-catalyzed reaction
Principle
〔 E 〕 T B C
velocity of enzyme-catalyzed reaction
〔 S 〕 A pH D E F
activator
inhibitor

4. Rectangular hyperbola

5. Michaelis-Menten equation
Km is Michaelis constant
When V= Vmax/2,
Km=[S]
the units of Km are the same as substrate concentration’s( mol/L)

6. Lineweaver-Burk double-reciprocal plot1 V = Km
Vmax ×
[S] +

7. pH10

8. Mixing , incubate in 37℃ water bath for 5 mins.
Protocol
1. Add reagents according to the following table.
(ml)
No. 1 2 3 4 5 6 7
Blank
0.02mol/L substrate
0.05
0.10
0.2
0.4
0.60
0.80
1.00
0.00
pH10 carbonate buffer
0.90
dH2O
0.95
0.40
0.20
Mixing , incubate in 37℃ water bath for 5 mins.
Enzyme (0.5mg/ml)
0.1
Mixing immediately, incubate in 37℃ water bath for 15mins exactly.。
NaOH
1.0
4-aminoantipyrene
K3Fe(CN)6
2.0
Mixing, place at room temperature for 10 mins, and record A510
A510 8

9. Conc. of substrate mmol/L
Calculation of data
Conc. of substrate mmol/L
0.50
1.0
2.0
4.0
6.0
8.0 10
1/[S] 2 1
0.5
0.25
0.167
0.125
0.10
1/ A510
Making a plot with 1/[S] as abscissa, 1/A510 as ordinate 9

10. According –1/Km, calculate the value of Km
Calculation
According –1/Km, calculate the value of Km
Double reciprocal graph

11. Demand 1. Two persons cooperate for the experiment
2. Enzyme solution used here is toxic, so carefully to handle it
3. The amount of E and S transferred should be exact
4. The incubation time should be exact as 15 minutes

12. Next week work To design a project about experiment research
To find the optimum pH for saliva amylase
How to do?
Which method shoul be used?
What is the principle?
Which kind of solution should be used?
What is concentration of a reagent?