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

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

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

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

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

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 1653-1660

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

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

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

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

HEPES Experiments 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

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

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

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

Pipes Experiments pH Pipes Concentration Pipes into HNO3 addition of Zn did not influence pH Pipes Experiments Pipes Concentration

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

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

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

BCS Experiments 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

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

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

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

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

Fit Data 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 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

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

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

The Journal of Biological Chemistry Vol. 286, NO. 13, pp 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.” Excel for Chemists: A Comprehensive Guide. E. Joseph Billo pp.349-351 Journal of Inorganic Biochemistry Vol. 99 Issue 8, August 2005, Pages 1653-1660 References

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

Questions?