Chelating Agents and
Temperature Effects on
Alkaline Phosphatase Activity
Steven Fan, Stefanie Lopez, Carrie Pusec, Jun Shi
Purpose

The purpose of the experiment is to use
activity assay to determine the effects of
chelating agents (EDTA and DTPA) and
the effects of temperature on Alkaline
phosphatase.
Alkaline Phosphatase and Metal ions
in Active Site
Zn2+
Mg2+
Metal ion roles in AP mechanism

Zn2+ ions most significant for
conformation stability and
catalytic function

roles in coordination of
nucleophilic attack and
facilitating departure of
leaving groups

Mg2+ unessential (Dieckmann)
but contributes to greater
stabilization and activity

generates hydroxyde for
serine deprotonation (Strec.
et al)
EDTA

six coordination
bonds with a metal
ion

general chelator
DTPA

eight coordination bonds
with metal ion

zinc specific chelator

formation constant
(equilibrium constant)
100 fold greater than
EDTA

relatively insoluble in
water, <0.5 g/ 100mL,
low pH ~ 2.5
Material and Methods
Performed experimental trials to determine the
[EDTA] and [DTPA] to use to make comparisons
with the temperature
 For each activity assay, absorbance was measured at
410nm with a spectrometer with 30 second intervals
with an extinction coefficient 0.0187 uM-1cm-1
 Five activity assays were performed

1.
2.
3.
4.
5.

AP with EDTA at RT
AP with EDTA at 50°C
AP with DTPA at RT with pH = 2.3
AP with DTPA at RT with pH = 8.4
AP with DTPA at 50°C pH = 2.3
Plotted V0 vs. Time to determine half-life based on its
exponential decay curve
Initial Conditions of AP/Chelator
Solution
EDTA
DTPA
Mixture
Concentrati
on mM
Mixture
Concentratio
n mM
EDTA
0.24
DTPA
0.005
Alkaline
Phosphatase
0.0073
Alkaline
Phosphatase
0.0073
Tris Buffer pH
7.4
10
Tris Buffer pH 10
7.4
Initial Conditions of the Activity
Assays for EDTA and DTPA
Assay Volume ( L)
AP/Chelator Solution
25
PNPP (0.658 mM)
500
Buffer (200 mM Tris pH 8.1)
500
Total
1025
EDTA
9
8
7
6
Vo
EDTA HIGH TEMP
5
EDTA ROOM TEMP
4
y = 6.9884e-0.041x
3
2
1
y = 5.8027e-0.272x
0
0
10
20
Time (min)
30
40
50
DTPA
8
DTPA HIGH
TEMP
7
DTPA ROOM
TEMP PH 8.4
6
Vo (uM/min)
5
DTPA ROOM
TEMP - PH 2.3
4
3
2
y = 2.9531e-0.052x
1
0
0
10
20
Time (min)
30
40
50
Half-Life Values for each Condition
Half-life (min)
EDTA Room Temperature
pH 8
11.23
EDTA Elevated Temperature 2.55
pH 8
DTPA Room Temperature
pH 2.3
Inconclusive (~hours)
DTPA Elevated Temperature 4.02
pH 2.3
General Conclusions
Generally, EDTA and DTPA at 25°C and
50°C showed an exponential decay
representing the enzyme activity with
respect to time.
 EDTA at the higher temperature increased
the enzyme activity and decreased the half
life by a factor of 4.4.
 DTPA is more temperature dependent than
EDTA because after heating the system, the
time it took for the exponential curve, the
half life of DTPA dropped from multiple
hours to about one hour

Conclusion Between Chelators

1.
2.
3.


The DTPA chelator has a bigger effect on the
inactivation of Alkaline Phosphatase
- Based on the facts….
DTPA binds 100 fold more strongly to the metal ions
than EDTA.
DTPA is an octadentate and EDTA is a hexadentate
Zinc metal is more essential for catalysis over
magnesium (Diekmann 2004).
The [DTPA] used was 50x less than the [EDTA].
The half life was DTPA at elevated temperature was
4.02 and the EDTA was 2.55, which is fairly close for
such a great difference in concentration.
Problems
The pH of DTPA
- pH influenced the solubility
- pH adjustment from 2.3 to around
neutral pH
 The concentration of EDTA was not
equal to DTPA to make a direct
comparison

References





Boguslaw Stec. 2000. A Revised Mechanism for the Alkaline
Phosphatase Reaction Involving Three Metal Ions. J. Mol. Biol.
1303-1311.
Coleman, JE. 1992. Structure and Mechanism of Alkaline
Phosphatase. Annu. Rev. Biophys. Biomol. Struct. 21:441-83.
Garen, A and C Levinthal. 1959. A Fine- Structure Genetic
and Chemical Study of the Enzyme Alkaline Phosphatase of E.
Coli. Biochem Biophys. Acta. 11;38:470-83.
Plocke DJ, Levinathal C, and BV Vallee. 1962. Alkaline
Phosphatase of Escherichia coli: Escherichia coli: A Zinc A
Zinc Metalloenzyme Metalloenzyme. . Biochem Biochem..
1;3:373 1;3:373—7
Plocke DJ, and BV Vallee. 1962. Interaction of Alkaline
Phosphatseof E. coli with Metal Ions and Chelating Agents.
Biochem. 1;6:1039-43
Meow its question time!
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