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 ChemistryBiochemistry 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

– 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.

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.

0 10 20 30 40 50 60 700

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Comparison of Enzyme Activity at 50º C

Control KCl NaClMgCl2

Time (min)

Enzy

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Activ

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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????????

0 10 20 30 40 50 60 700

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Comparison of Enzyme Activity at 60ºC

ControlKClNaClMgCl2

Time (min)

Enzy

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Results:

Due to the effects of the Hofmeister Series, over time, MgCl2 displayed kosmotropic characteristics.

0 10 20 30 40 50 60 700

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140Comparison of Enzyme Activity at 70ºC

ControlKClNaClMgCl2

Time (min)

Enzy

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Activ

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Results:

Throughout the course of thermal incubation of AP, NaCl and KCl showed similar effects on enzyme activity as depicted by the Hofmeister Series.

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140NaCl and KCl Comparison at 50º C

KCl 50 NaCl 50

Time (min)

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NaCl and KCl Comparison at 60º C

KCl 60 NaCl 60

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NaCl and KCl Comparison at 70º C

KCl 70 NaCl 70

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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.

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Temperature Comparison of MgCl2

MgCl2 50MgCl2 60MgCl2 70

Time (min)

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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 821-828

• 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