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

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Presentation on theme: "Determination of Metal Binding Constants by Potentiometric Titrations Presentation by: Destinee K. Johnson Research Mentor: Dr. Grossoehme."— Presentation transcript:

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 ITC Data Background of Zinc titrated into EDTA in HEPES buffer

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

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

7 Buffer: Pipes PipesPipes-Zn Complex

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

9 Hypothesis 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 + + Zn 2+ B-Zn 2+

10 Procedure 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.

11 HEPES Experiments HEPES in HNO 3 …with Zn present Titration of 50 mL of 5 mM HNO 3, 100 mM KNO 3 with mM HEPES, 100 mM KNO 3 Titration of 50 mL of 5 mM Zn, 5 mM HNO 3, and 100 mM KNO 3 with mM HEPES, 100 mM KNO 3

12 HEPES Experiments HEPES Concentration pH

13 HEPES Experiments HNO 3 in HEPES …with Zn present Titration of 50 mL of 5 mM HEPES and 100 mM KNO 3 with 5 mM HNO 3 Titration of 50 mL of 5 mM Zn, 5 mM HEPES, 100 mM KNO 3 with 5 mM HNO 3

14 Pipes Experiments Pipes in HNO 3 …with Zn present Titration of 50 mL of 5 mM HNO 3 and 100 mM KNO 3 with 50 mM Pipes and 100 mM KNO 3 Titration of 50 mL of 5 mM Zn, 5 mM HNO 3 and 100 mM KNO 3 with 50 mM Pipes and 100 mM KNO 3

15 Pipes Experiments Pipes Concentration pH

16 Pipes Experiments HNO 3 in Pipes …with Zn present Titration of 50 mL of 5 mM Pipes and 100 mM KNO 3 with 50 mM HNO 3 and 100 mM KNO 3 Titration of 50 mL of 5 mM Zn, 5 mM Pipes and 100 mM KNO 3 with 50 mM HNO 3 and 100 mM KNO 3

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

18 Other Experiments Determination of formation constants in the Cu 2+ -BCS system Determination of formation constants in the Cu + -BCS system with Cu + stabilized in acetonitrile

19 BCS Experiments HNO 3 in BCS …with Cu 2+ present Titration of 25 mL of.6255 mM BCS with mM HNO 3 Titration of 25 mL of.31 mM Cu(II) and.6255 mM BCS with mM HNO 3

20 BCS Experiments

21 Stability Constants -The constant K 1 is called a stability constant M 2+ + L - ML +

22 Metal Complex Formation M 2+ + L - ML + ML + + L - ML 2 β 1 = K 1 β 2 = K 1 K 2

23 Bjerrum Method 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)

24 Fit Data Fit parameters: Y-int 2.07 ± 0.06 Slope 0.17 ± 0.07 K 1 = 2.07 ± 0.06 x 10 6 β 2 = 0.17 ± 0.07 x Credits: Dr. Nicholas Grossoehme

25 Actual Expected BCS-Cu + Experiments Titration of 25 mL of.4 mM Cu(I), 50 mM MeCN, and.66 mM BCS with 5 mM HNO 3

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

27 References The Journal of Biological Chemistry Vol.286, NO.13, pp , April 1, 2011 Synthesis and Technique in Inorganic Chemistry Third Edition, pp Quinn, Colette.Analyzing ITC Data for the Enthalpy of Binding Metal Ions to Ligands. Calculation of Binding Constants. Excel for Chemists: A Comprehensive Guide. E. Joseph Billo pp Journal of Inorganic Biochemistry Vol. 99 Issue 8, August 2005, Pages

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

29 Questions?


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