1. 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

2. 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.

3. 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)”.

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 Mg 2+ salts) would propagate an increase in the catalytic performance of the enzyme.

5. 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 Mg 2+ salt would show the least effect on enzyme activity over the course of thermal incubation.

6. 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 MgCl 2 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.

7. 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 MgCl 2 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 MgCl 2 – KCl- Sigma Chemical Company (Available in stock room) – NaCl- Available in Dr. Hazzard’s stock room – MgCl 2 - Sigma Chemical Company (Available in stock room)

8. 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 MgCl 2 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.

9. 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 MgCl 2 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.

10. 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.

11. Results: The buffer containing the Mg 2+ 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????????

12. Results: Due to the effects of the Hofmeister Series, over time, MgCl 2 displayed kosmotropic characteristics.

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

14. 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.

15. 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.

16. 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