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

2. EDTA Ethylenediaminetetraacetic acid- polyamino carboxylic acid
EDTA is a weak base and in aqueous solution it can be protonated (Bronsted-Lowry)
EDTA

3. EDTA Hexadentate ligand-has six sites that embrace the zinc ion (4 oxygen and 2 nitrogen)
The EDTA-Zn complex formation involves competition between the proton and Zn for EDTA
EDTA-Zn Complex

4. Background of Zinc titrated into EDTA in HEPES buffer
ITC (Isothermal Titration Calorimetry) measures the enthalpy change for a reaction. One of the major heat producing events in a reaction can be related to the displacement of protons from a binding ligand or METAL LIGAND COMPLEX FORMATION
Protons displaced from EDTA when Zn binds after this we expect the dilution to be small but unexpectedely it is very big after EDTA has already been saturated. This means that this large curve is because of an interaction between Zn and the Buffer not between Zn and EDTA
Large and unexpected dilution
*REGION SHOWS ZINC BINDING TO EDTA AND OTHER COUPLED REACTIONS (HEAT ASSOCIATED WITH) THESE REACTIONS
ITC Data

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

6. Buffer: HEPES HEPES HEPES-Zn Complex
Rorabacher proposed that Zn binds to oxygen and toenails it way to the nitrogen displacing the proton
Buffer: HEPES
Rorabacher, D.B. Journal of Inorganic Biochemistry Vol. 99 Issue 8, August 2005, Pages

7. Buffer: Pipes Pipes Pipes-Zn Complex Pka near 6.76 physiological Ph
In the pipes complex we THINK zn just binds to the 2 nitrogrens
Buffer: Pipes

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
Other methods include NMR and spectrometry but Zn is a spectroscopically metal because of the d-shell so those methods cannot be used
The metal-ligand complex formation involves competion between the proton and metal ion for the ligand base
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+
****the presence of the metal ion influences pH Zn binds to the same place a proton would proton is displaced and solution becomes extremely acidic ….hence complex formation
Zn bond inhibits protonation (spend A LOT of time on THIS slide)
Coupled equilibria
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.
Ionic salt used in experiment was potassium nitrate. This eliminates uncertainties arising from activity constant coefficient variations.
To keep ionic strength constant unreactive ionic salt is added to maintain the ionic strength of the medium and does not interact with the metal or ligand.
Use volumetric flasks, calibrated pipets, milli-q water, ph meter
Procedure

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

12. HEPES Experiments pH HEPES Concentration
Addition of Zn did not influence pH (HEPES into HNO3)
HEPES Experiments
HEPES Concentration

13. HEPES Experiments HNO3 in HEPES …with Zn present
REVERSE DIRECTION OF PREVIOUS EXPERIMENT
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 Experiments 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. Pipes Experiments pH Pipes Concentration
Pipes into HNO3 addition of Zn did not influence pH
Pipes Experiments
Pipes Concentration

16. Pipes Experiments HNO3 in Pipes …with Zn present
Reverse direction of previous experiment
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
The pH is not influenced when Zn is present
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
The pH titrations helped me develop the technique to determine the binding affinity of Copper (I) and BCS
By stabilizing copper in acetonitrile we can prevent disproportionation and determine the elusive k1 value for bcs and copper
Other Experiments

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

20. Green and black- at the equivalence where ph equals pka pka of one of the nitrogens in bcs. One of the nitrogens are being protonated
Red and blue- ½ molar equivalency of copper 2 bcs to 1 cu2+
BCS Experiments

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

22. Metal Complex Formation
M2+ + L ML+
ML+ + L ML2
β1 = K1
β2 = K1K2
Bathocuproine disulfonate structure
two equilibria possible
The equilbria constants can also be expressed as overall stability constants which are simply products of the stepwise stability constants
Metal Complex Formation

23. Θ, the average number of ligand molecules bound per metal ion
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)
Methods for calculating stability constants: To determine the K values a function nbar (theta) is defined as the average number of ligand molecules bound per metal ion (bjerrum method)
Use values that we know to determine the stability constant
From an experimental stand point n bar may be expressed in terms of total copper or metal ion concentration
1:1 Cu BCS
1:2 Cu(BCS)2 – equilbrium math
Bjerrum Method

24. Fit Data Fit parameters: Y-int  2.07 ± 0.06 Slope  0.17 ± 0.07
K1 = 2.07 ± 0.06 x 106
β2 = ± 0.07 x 1011
The original equation was converted to this by introducting expressions fro the overall stability constants, Beta where b1= K1 b2=K1K2 and rearrange to y=mx+b
Plot to determine the overall stability constant k1 value
This plot tends to a straight line at low concentration of bcs in basic from w/o proton or metal ion bound of intercept K1 and slope beta2
Fit Data
Credits: Dr. Nicholas Grossoehme

25. BCS-Cu+ Experiments Actual Expected
Under anaerobic conditions in glove box!
Cu (1) stabilized in MeCN
**NO DOUBT THAT WE FORMED A COMPLEX BC OF THE DEEP ORANGE COLOR BUT SOMETHING WAS ASMISS IN DATA AND WE DIDN’T HAVE TIME OR MATERIALS TO REPEAT EXPERIMENT
Expect a faster decline (blue line)
Solution was a deep red color
Determine K1 value for Cu(1) BCS by stabilizing Cu(1) in MeCN
Titration of 25 mL of .4 mM Cu(I), 50 mM MeCN, and .66 mM BCS with 5 mM HNO3
BCS-Cu+ Experiments

26. Prospects for the Future
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
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
References

28. Acknowledgements The Grossoehme Lab
Winthrop University Chemistry Department
Winthrop University Summer Undergraduate Research Experience
Idea Network for Biomedical Research Excellence
Dr. Nicholas Grossoehme, Sharon Jenkins, Paisley Trantham, Zayed Almadidy, Becca Toor
Acknowledgements

29. Questions?