Chelating Agents and Temperature Effects on Alkaline Phosphatase Activity

Steven Fan, Stefanie Lopez, Carrie Pusec, Jun Shi

PurposeThe 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

Mg2

+

Zn2

+

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)

EDTAsix coordination

bonds with a metal ion

general chelator

DTPAeight 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 MethodsPerformed 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 performed1. AP with EDTA at RT2. AP with EDTA at 50°C3. AP with DTPA at RT with pH = 2.3 4. AP with DTPA at RT with pH = 8.45. 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 Concentration mM

EDTA 0.24

Alkaline Phosphatase

0.0073

Tris Buffer pH 7.4

10

Mixture Concentration mM

DTPA 0.005

Alkaline Phosphatase

0.0073

Tris Buffer pH 7.4

10

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

0 5 10 15 20 25 30 35 40 450

1

2

3

4

5

6

7

8

9

f(x) = 6.98835794417777 exp( − 0.0413352207563553 x )

f(x) = NaN exp( NaN x )

EDTA HIGH TEMP

Exponential (EDTA HIGH TEMP)

EDTA ROOM TEMP

Logarithmic (EDTA ROOM TEMP)

Exponential (EDTA ROOM TEMP)

Time (min)

Vo (uM

/m

in)

DTPA

0 5 10 15 20 25 30 35 40 450

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

f(x) = NaN exp( NaN x )

DTPA HIGH TEMP

Exponential (DTPA HIGH TEMP)

Time (min)

Vo

(uM

/min

)

Half-Life Values for each Condition

Half-life (min)

EDTA Room TemperaturepH 8

11.23

EDTA Elevated TemperaturepH 8

2.55

DTPA Room TemperaturepH 2.3

Inconclusive (~hours)

DTPA Elevated TemperaturepH 2.3

4.02

General ConclusionsGenerally, 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

The DTPA chelator has a bigger effect on the inactivation of Alkaline Phosphatase

- Based on the facts….1. DTPA binds 100 fold more strongly to the metal

ions than EDTA. 2. DTPA is an octadentate and EDTA is a

hexadentate3. 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.

ProblemsThe pH of DTPA - pH influenced the solubility - pH adjustment from 2.3 to around neutral pHThe 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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