Determination of Metal Binding Constants by Potentiometric Titrations

1. Determination of Metal Binding Constants by Potentiometric Titrations Presentation by: Destinee K. Johnson Research Mentor: Dr. Grossoehme

2. EDTA

3. EDTA-Zn Complex

4. Background of Zinc titrated into EDTA in HEPES buffer ITC Data

5. To use potentiometric titrations to extract the metal binding constants of Zn for various buffers Goal of Project

6. HEPES HEPES-Zn Complex Buffer: HEPES Rorabacher, D.B. Journal of Inorganic Biochemistry Vol. 99 Issue 8, August 2005, Pages 1653-1660

7. Pipes Pipes-Zn Complex Buffer: Pipes

8. Method:Potentiometry • Why? -Zinc is a spectroscopically silent metal -The progress of the metal-ligand complex formation can be monitored by pH measurements Calculation of Binding Constants

9. It will be more difficult for protons to bind to the ligand (HEPES/PIPES buffer) in the presence of Zn. This competition will influence the pH. B + H+ BH+ + Zn2+ B-Zn2+ Hypothesis

10. Appropriate volumes of ligand solution, metal-ion solution, ionic salt, and metal free water are transferred into a beaker. • The solution is stirred magnetically. • Increments of titrant are added and the pH is recorded using a pH meter after the addition of each increment. Procedure

11. HEPES in HNO3 • …with Zn present Titration of 50 mL of 5 mM HNO3 , 100 mM KNO3 with 39.899 mM HEPES, 100 mM KNO3 Titration of 50 mL of 5 mM Zn, 5 mM HNO3 , and 100 mM KNO3 with 39.899 mM HEPES, 100 mM KNO3 HEPES Experiments

12. pH HEPES Experiments HEPES Concentration

13. HNO3 in HEPES • …with Zn present Titration of 50 mL of 5 mM HEPES and 100 mM KNO3 with 5 mM HNO3 Titration of 50 mL of 5 mM Zn, 5 mM HEPES, 100 mM KNO3 with 5 mM HNO3 HEPES Experiments

14. Pipes in HNO3 • …with Zn present Titration of 50 mL of 5 mM HNO3 and 100 mM KNO3 with 50 mM Pipes and 100 mM KNO3 Titration of 50 mL of 5 mM Zn, 5 mM HNO3 and 100 mM KNO3 with 50 mM Pipes and 100 mM KNO3 Pipes Experiments

15. pH Pipes Experiments Pipes Concentration

16. HNO3 in Pipes • …with Zn present Titration of 50 mL of 5 mM Pipes and 100 mM KNO3 with 50 mM HNO3 and 100 mM KNO3 Titration of 50 mL of 5 mM Zn, 5 mM Pipes and 100 mM KNO3 with 50 mM HNO3 and 100 mM KNO3 Pipes Experiments

17. A metal-ligand complex did not form between Zn and Pipes or HEPES at the working concentrations • Zinc becomes diluted Conclusion

18. Determination of formation constants in the Cu2+ -BCS system • Determination of formation constants in the Cu+-BCS system with Cu+ stabilized in acetonitrile Other Experiments

19. HNO3 in BCS • …with Cu2+present Titration of 25 mL of .6255 mM BCS with 6.1328 mM HNO3 Titration of 25 mL of .31 mM Cu(II) and .6255 mM BCS with 6.1328 mM HNO3 BCS Experiments

20. BCS Experiments

21. M2+ + L- ML+ -The constant K1 is called a stability constant Stability Constants

22. M2+ + L- ML+ ML+ + L- ML2 β1 = K1 β2 = K1K2 Metal Complex Formation

23. From pH measurements and knowledge of quantities originally added it is possible to calculate the stability constants Θ, the average number of ligand molecules bound per metal ion Cu + 2BCS Cu(BCS)2 Cu + BCS Cu(BCS) Bjerrum Method

24. Fit parameters: Y-int  2.07 ± 0.06 Slope  0.17 ± 0.07 K1 = 2.07 ± 0.06 x 106 β2 = 0.17 ± 0.07 x 1011 Fit Data Credits: Dr. Nicholas Grossoehme

25. Actual Expected Titration of 25 mL of .4 mM Cu(I), 50 mM MeCN, and .66 mM BCS with 5 mM HNO3 BCS-Cu+ Experiments

26. Develop a Cu+ stabilizing system to investigate the thermodynamic properties that control Cu+ binding and selectivity. Prospects for the Future

27. The Journal of Biological Chemistry Vol.286, NO.13, pp. 11047-11055, April 1, 2011 • Synthesis and Technique in Inorganic Chemistry Third Edition, pp. 219-231 • Quinn, Colette.“Analyzing ITC Data for the Enthalpy of Binding Metal Ions to Ligands.” • “Calculation of Binding Constants.” Excelfor Chemists: A Comprehensive Guide. E. Joseph Billo pp.349-351 • Journal of Inorganic Biochemistry Vol. 99 Issue 8, August 2005, Pages 1653-1660 References

28. The Grossoehme Lab Winthrop University Chemistry Department Winthrop University Summer Undergraduate Research Experience Idea Network for Biomedical Research Excellence Acknowledgements

29. Questions?