Determining the Zinc and Magnesium
Dependencies of Alkaline Phosphatase
Andrew Ma, Elina Ly, Audrey Shi, and
Ashley Vergara
BIOC 463a
Tuesday, November 22, 2011
Alkaline Phosphatase
• Hydrolase enzyme found in E. coli
• Located in the periplasm
• Responsible for removing
phosphate groups from various
chemicals
• Expression increases in the
absence of phosphate
• Highly resistant to thermal
inactivation and denaturation
• Exists as a dimer, each one
containing two zinc and one
magnesium ions
• Optimal activity at pH = 8.0
1. Stec, B., et al. (2000) “A revised mechanism for the alkaline phosphatase reaction involving three metal ions.” J. Mol. Biol., 299, 1303-1311.
2. Garen, A. and Levinthal, C. (1959). “A Fine-Structure Genetic and Chemical Study of the Enzyme Alkaline Phosphatase of E. Coli.” Biochem.
Biophys Acta., 38, 483-494.
The Effects of Chelation
• Chelating agents are chemical
compounds that bind certain
metals by forming coordination
bonds with the metal ions
• EDTA is a common chelator
that forms 6 coordination bonds
• DTPA is a stronger chelator that
forms up to 8 coordination
bonds
• Chelating agents can remove
metal ions from proteins and
affect their overall activity
EDTA, a common chelator for many metals
DTPA, a stronger chelator for metal ions
Plock, D.J., and Vallee, B.L. (1962). “Interaction of Alkaline Phosphatase of E. coli with Metal Ions and Chelating Agents.” Biochemistry, 1,
1039-1043.
Experimental Purpose and Design
• To determine how AP activity is affected when treated with the
chelating agent DTPA and how it compares to treatment with EDTA
• Monitor effects by conducting activity assays and recording initial
velocity (Vo) readings for the following:
*AP treated with EDTA
-Incubation performed at room
temperature
-Varying incubation time
*AP treated with DTPA
-Incubation performed at room
temperature
-Varying incubation time
-Varying concentrations
Enzyme Preparation
• Diluted 30 uL of purchased AP
into 1 mL of Tris buffer, pH = 7.4.
• The dilution was then added to a
centricon.
Alkaline
Phosphatase
Tris
pH = 7.4
• Dialyzed the AP with Tris buffer, pH = 7.4 to
remove various metal-containing salts.
• Dialysis was performed in a 50 kDA Amicon
Centricon, which also concentrated the
enzyme within a smaller volume
Assay Protocol
AP Inactivation Mix
15 uL of 0.1 M
chelating agent
150 uL of 30
uM AP
Incubate inactivation mix
at room temperature
Kinetics Assay Mix
25 uL
inactivation mix
450 uL Tris, pH = 8.0
500 uL 1.2 mM PNPP
Read activity at 410 nm
and record Vo
Results
7
6
10 mM EDTA
Vo (uM/min)
5
10 mM DTPA
5 mM DTPA
4
3
2
y = 5.9062e-0.059x
1
y = 5.3139e-0.067x
y = 5.0825e-0.122x
0
0
5
Time at which activity
is halved:
5.68 min.
10
15
Time (min)
20
25
10.35 min. 11.75 min.
Each curve represents the average of two inactivation assays.
30
Conclusion
• Incubation with DTPA is an effective method
for inactivating alkaline phosphatase activity
• DTPA is a chelating agent that inactivates AP
nearly twice as quickly as does EDTA
• DTPA could possibly be used for other assays
in which alkaline phosphatase activity needs
to be inhibited
Future Directions
• Determining the pH, temperature, and
concentration dependencies of AP inactivation
via metal chelation
• Investigate whether only zinc ions are
removed or if magnesium is also removed
• Observing AP’s activity when it uses other
metal cofactors after treatment with DTPA
References
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•
•
•
•
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Coleman, J.E. (1992). “Structure and Mechanism of Alkaline Phosphatase.” Annu. Rev. Biophys. Biomol. Struct.,
21, 441-83.
Garen, A. and Levinthal, C. (1959). “A Fine-Structure Genetic and Chemical Study of the Enzyme Alkaline
Phosphatase of E. Coli.” Biochem. Biophys Acta., 38, 483-494.
Kim, E.E. and Wyckoff, H.W. (1991) “Reaction mechanism of alkaline phosphatase based on crystal structures.
Two-metal ion catalysis.” J. Mol. Biol., 218, 449-464.
Ninfa, Alexander J, Ballou, David P., and Marilee Benore. “Fundamental Laboratory Approaches for Biochemistry
and Biotechnology, Second Edition.” John Wiley & Sons, Inc.: 2010.
Plock, D.J., and Vallee, B.L. (1962). “Interaction of Alkaline Phosphatase of E. coli with Metal Ions and Chelating
Agents.” Biochemistry, 1, 1039-1043.
Stec, B, Holtz, K.M., and Kantrowitz, E.R. (2000). “A Revised Mechanism for the Alkaline Phosphatase Reaction
Involving Three Metal Ions.” J. Molecular Biology, 299, 1303-1311.