Transcript Document

The Effects of Different Cationic
Salts on the Thermal Stability of
Alkaline Phosphatase
Authors: Nancy Leo, Nicholas Nelson,
David Wasiak, Sara Zarr
University of Arizona Department of Biochemistry and Chemistry
Biochemistry 463A- Dr. James Hazzard
22 November, 2011
INTRODUCTION
• Alkaline Phosphatase (AP) demonstrates
maximum activity in an environment with an
approximate pH of 8.0 (Boguslaw Stec 1).
• In his paper, Yang et. al highlights that AP
exhibits its optimal activity in the presence of
salt ions with similar positions on the scale of
the Hofmeister series.
• He indicates that AP activity was maximized
with salts such as NaCl and KCl.
HOFMEISTER SERIES
“ Proteins are usually found to be stabilized by a kosmotropic anion and a
chaotropic cation and destabilized by a chaotropic anion and a kosmotropic
cation (Yang et. al. 4)”.
GOALS
• The goals of this experiment were to
determine which cationic salt—present in the
buffer containing AP—stabilizes the enzyme
during a period of thermal incubation.
• We were interested in analyzing which cation
(from a series of K+, Na+, and Mg2+ salts)
would propagate an increase in the catalytic
performance of the enzyme.
HYPOTHESIS
• According to the Hofmeister Series, we expected
that the buffers containing the K+ and Na+ cations
would stabilize AP, and lead to increased enzyme
activity (thus increasing the thermal stability of
the enzyme) over the course of thermal
incubation.
• Additionally, we expected the AP exposed to the
buffer containing the Mg2+ salt would show the
least effect on enzyme activity over the course of
thermal incubation.
METHODS AND MATERIALS
• In order to test which cationic salts attributed
to the greatest increase in thermal stability of
AP, a control buffer of 10mM Tris-HCl, and
three salt buffers of 1M KCl, NaCl, and MgCl2
were prepared.
• Preparation of 10mM Tris-HCl buffer
– 2 mL of 200 mM HCl , 2 mL of 200mM NaOH and
76 mL of reagent grade water were added to a
beaker to produce a final solution of 80 mL 10mM
Tris-HCl.
METHODS AND MATERIALS cont.
• Preparation of 1M Salt Solutions in 10 mM Tris-HCl buffer
– 1.11825 g of KCl, 0.7419 g of NaCl and 3.0495 g of MgCl2 were
weighed out, and the solid salts were placed individually into three
different 15 mL conical tubes. All tubes were labeled and a control
tube was filled with 15 mL of 10 mM Tris-HCl buffer.
– 10mM Tris-HCl buffer was added to each of the tubes containing the
solid salts, and the salts were allowed to dissolve. Once dissolved, the
tubes were vortexed to homogenize the solution and three final
solution of 15 mL, 1M salt solutions in 10 mM Tris-HCl.
– Each of the 15 mL salt buffers and control were aliquoted into 3
separate tubes, each of 5 mL total volume (for 50, 60, and 70
temperature experiments).
– The pH of the salt buffer solutions were adjusted and maintained at a
pH of 8.0 utilizing a small quantity of 1M NaOH.
• Stock Manufacturer for KCl, NaCl and MgCl2
– KCl- Sigma Chemical Company (Available in stock room)
– NaCl- Available in Dr. Hazzard’s stock room
– MgCl2- Sigma Chemical Company (Available in stock room)
METHODS AND MATERIALS cont.
• Preparation of 0.658mM PNPP solution
– 0.03g of dried PNPP was dissolved in 100 mL of reagent
grade water.
• Preparation of 3 uM AP Enzyme
– 200 U of AP were obtained from a Sigma Chemical
Company bottle.
– 1 mL of 5 mM Tris buffer (pH 7.4) and 5 mM MgCl2 was
added to the lyophilized powder containing protein and
buffer salts, and the solution was allowed to completely
dissolve.
– To perform enzyme steady state kinetics, 30 uL of this
solution—after the protein was completely dissolved—was
added to 1 mL of the same buffer to prepare a 3uM AP
enzyme solution.
METHODS AND MATERIALS cont.
•
Experimental Protocol
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A volume of 1100 uL was extracted from each of the 5 mL control and salt buffer solutions; this
volume was placed in an 2 mL Epindorf tube. A total of 4 Epindorf tubes, per temperature condition,
were made.
The water bath was heated to a temperature of 50° C.
A cuvette containing 450 uL of 0.658 mM PNPP and 450 uL of 10 mM Tris-HCl was utilized to zero the
Enzyme Kinetics program (using the Cary 50).
The program was set up to read at 410 nm, for a period of 0-0.5 min, and with an extinction
coefficient of 0.01833 M-1 .
50 uL of 3mM AP enzyme solution was added simultaneously to the Control and KCl Epindorf tube.
The tubes were mixed and 100uL of the solutions were removed and dispensed into the cuvette
containing both PNPP and Tris-HCL buffer.
The initial velocity of enzyme activity was determined in uM/min from the slope analyzer.
The control and KCl tubes were immediately placed into the water bath, and 100 uL of the samples
were removed at 6 min intervals and initial velocity was determined over a 60 minute time interval.
• New PNPP and buffer were added to the cuvette before the successive addition of the 100 uL
aliquots.
The above procedure was repeated for the MgCl2 and NaCl pair 2 minutes after the control and KCl
tubes were placed into the water bath (these tubes were off-set from the first pair by 2 minutes).
The above protocol was repeated for the 60° and 70 ° samples as well.
Comparison of Enzyme Activity at 50º C
140
120
Enzyme Activity (U)
100
80
Control
KCl
60
NaCl
MgCl2
40
20
0
0
10
20
30
40
50
60
70
Time (min)
Results:
Thermal stability of Alkaline Phosphatase (AP) was shown to
increase in the presence of cations in the buffer solution
compared to the control of Tris-HCl buffer alone.
Comparison of Enzyme Activity at 60ºC
140
120
Enzyme Activity (U)
100
80
Control
KCl
60
NaCl
MgCl2
40
20
0
0
10
20
30
40
50
60
70
Time (min)
Results:
The buffer containing the Mg2+ cation promoted greater enzyme activity compared to the buffers containing
Na+ and K+ cations.
This contradicts magnesium’s relative
position in the Hofmeister Series
**THERE MUST BE A REASON WHY AP EXPOSED TO THE MG SALT BUFFER EXHIBITS SUCH AN INCREASE IN
ENZYME ACTIVITY????????
Comparison of Enzyme Activity at 70ºC
140
120
Enzyme Activity (U)
100
80
Control
KCl
NaCl
60
MgCl2
40
20
0
0
10
20
30
40
50
60
70
Time (min)
Results:
Due to the effects of the Hofmeister Series, over time, MgCl2
displayed kosmotropic characteristics.
NaCl and KCl Comparison at 60º C
NaCl and KCl Comparison at 50º C
140
100
90
120
80
KCl 50
60
NaCl 50
40
Enzyme Activity (U)
Enzyme Activity (U)
80
100
70
60
KCl 60
50
NaCl 60
40
30
20
20
10
0
0
20
40
60
0
80
0
20
Time (min)
60
80
Time (min)
NaCl and KCl Comparison at 70º C
Results:
100
90
80
Enzyme Activity (U)
Throughout the course of
thermal incubation of AP, NaCl
and KCl showed similar effects on
enzyme activity as depicted by
the Hofmeister Series.
40
70
60
50
KCl 70
40
NaCl 70
30
20
10
0
0
20
40
Time (min)
60
80
Temperature Comparison of MgCl2
160
140
Enzyme Activity (U)
120
100
MgCl2 50
80
MgCl2 60
60
MgCl2 70
40
20
0
0
10
20
30
40
50
60
70
Time (min)
Results:
At increased temperatures, Mg is primarily acting as a kosmotrope,
destabilizing the protein. This decreases enzyme activity, instead of
acting as a beneficial external cofactor in the catalytic mechanism of AP.
CONCLUSIONS
• Over the course of thermal incubation, Na+ and K+ showed
to have similar affects on enzyme activity, delineating
similar kosmotropic/chaotropic characteristics of the
KCl/NaCl cation-anion pair.
• At higher temperatures of incubation magnesium showed
characteristics of a destabilizing kosmotrope as described
by the Hofmeister Series.
• At lower temperatures, however, magnesium displayed a
greater increase in enzyme activity compared to sodium
and potassium which can be described by its mediation of
transphosphorylation through coordination with a water
ligand, stabilizing the phosphoryl group.
REFERENCES
• Garen A., Levinthal C. A fine-structure genetic and chemical study of
the enzyme alkaline phosphatase of E. coli. I. Purification and
characterization of alkaline phosphatase. Biochim Biophys Acta.
1960 Mar 11;38:470-83.
• Stec B, Holtz KM, Kantrowitz ER. A revised mechanism for the
alkaline phosphatase reaction involving three metal ions. J Mol Biol.
2000 Jun 23;299(5):1303-11.
• Yang,Liu,Chen,Halling, Hofmeister effects on activity and stability of
alkaline phosphatase, Biochimica et Biophysica Acta (BBA) - Proteins
& Proteomics, Volume 1804, Issue 4, April 2010, Pages 821828
• Zalatan, Fenn, Herschlag, Comparative Enzymology in the Alkaline
Phosphatase Superfamily to Determine the Catalytic Role of an
Active-Site Metal Ion, Journal of Molecular Biology, Volume 384,
Issue 5, 31 December 2008, Pages 1174-1189