1. Measuring Ion Binding of Proteins in Aqueous Media Shelby Bickford Dr. V. G. J. Rodgers Department of Bioengineering University of California, Riverside VGJR-The B2K Group | BIOTRANSPORT & BIOREACTION KINETICS

2. Introduction This study will describe ion binding as a function of varying pH, ionic strength, and protein concentration. The research will also determine the level of ionic concentration necessary for ion binding of proteins to become saturated. This data will be gathered for the binding of sodium and chloride ions by four globular proteins.

3. Background Ion binding of proteins has been studied for ~100 years. Ion binding of proteins has been studied for ~100 years. Example: Klotz I, Walker M, Pivan R The Binding of Organic Ions by Proteins. 1946 Ion binding of proteins is a vital biological activity. Ion binding of proteins is a vital biological activity. Example: Oxygen binding of Hemoglobin A better understanding of ion binding of proteins has practical applications in many fields. A better understanding of ion binding of proteins has practical applications in many fields. Example: Yamaguchi A, Tamang DG, Saier MH Mercury transport in bacteria Mercury transport in bacteriahttp://www.psc.edu/~deerfiel/NIH/Protein-Calcium_Ion-Site_Thumb.gif

4. Background Bovine Serum Albumin (BSA) – responsible for shuttling fatty acids, and other water insoluble molecules through the body. Bovine Alpha-Lactalbumin – an important whey protein found in cow’s, and other mammals, milk. Hen Egg Lysozyme (HEL) – can break down bacterial cell walls and therefore help white blood cells engulf bacteria. Bovine Cytochrome-c – an electron donor in the electron transfer chain of the mitochondria. It also aids in apoptosis. mgl.scripps.edu/.../pdb/pdb37/pdb37_1.html www.strubi.ox.ac.uk/.../crystallography.html www.bath.ac.uk/~bsssi/ravi/lactal2.htmlwww.bio.davidson.edu/.../yeastproteins.html

5. Background Salt is naturally present in the body. Salt is naturally present in the body. It helps maintain the electrolyte balance inside and outside the cells. Knowing the effect naturally occurring salt has on protein binding is advantageous.

6. Techniques for Measuring Ion Binding Dialysis Method – measures ion binding by either conductivity, difference UV spectroscopy, or collodion membrane electrodes. Dialysis Method – measures ion binding by either conductivity, difference UV spectroscopy, or collodion membrane electrodes. www.kbac-hi.org/BMPs/content/def_conductivity.htm www.chemicool.com/definition/spectronic_20.html www.kbac-hi.org/BMPs/content/def_conductivity.htm www.chemicool.com/definition/spectronic_20.html http://www.science.uwaterloo.ca/~cchieh/cact/c123/emf.html http://www.science.uwaterloo.ca/~cchieh/cact/c123/emf.html Electromotive Force Electromotive Force Cell Method

7. Previous Studies Irving M. Klotz, F. Marian Walker, and Rita B. Pivan Irving M. Klotz, F. Marian Walker, and Rita B. Pivan The Binding of Organic Ions by Proteins. 1946 G. Scatchard, I. H. Scheinberg, and S. H. Armstrong, Jr. G. Scatchard, I. H. Scheinberg, and S. H. Armstrong, Jr. Physical Chemistry of Protein Solutions. IV. The Combination of Human Serum Albumin with Chloride Ion. 1950

8. Previous Studies C. Carr Studies on the Binding of Small Ions in Protein Solutions with the Use of Membrane Electrodes. III. The Binding of Chloride Ions in Solutions of Various Proteins. 1953 W. Harris, A. Cafferty, S. Abdollahi, And K. Trankler Binding of Monovalent Anions to Human Serum Transferrin. 1997

9. Methods and Materials Dialysis Method using conductivity as a measure of ionic strength. Dialysis Method using conductivity as a measure of ionic strength. 0.5 ml of 0.001M BSA solution was injected into the dialysis cassette. 0.5 ml of 0.001M BSA solution was injected into the dialysis cassette. 3 cassettes were placed into a water-tight bag containing 3 ml of NaCl solution: 0.15 M, 0.1 M, 0.01 M, or 0.001 M. 3 cassettes were placed into a water-tight bag containing 3 ml of NaCl solution: 0.15 M, 0.1 M, 0.01 M, or 0.001 M. Bags were used as opposed to beakers so that a low volume of solution could be used. Bags were used as opposed to beakers so that a low volume of solution could be used.

10. Methods and Materials Each NaCl solution is assessed with an Orion conductivity probe, an Orion Chloride Ion Selective Electrode (ISE), and a pH meter before the cassettes are added. Each NaCl solution is assessed with an Orion conductivity probe, an Orion Chloride Ion Selective Electrode (ISE), and a pH meter before the cassettes are added. Once equilibrium is reached the solution is analyzed again. Once equilibrium is reached the solution is analyzed again. The difference in conductivity allows the number of bound ions to be calculated. The difference in conductivity allows the number of bound ions to be calculated. Orion Conductivity Probe Orion ISE Probe Orion pH Meter

11. Conductivity

13. Conductivity Calculations for NaCl Infinite Dilution 0.0005Molarity 0.001 Molarity 0.005 Molarity 0.01 Molarity 0.02 Molarity 0.05 Molarity 0.1 Molarity 126.39124.44123.68120.59118.45115.70111.01106.69 Theoretical Values of conductivity given in at a temperature of 25° C. Concentration is in mol/L. Na+ 50.08 at infinite dilution Cl- 76.31 at infinite dilution Concentration Conductivity

14. Electroneutrality Solution Chamber [ II ] Solution Chamber [ I ] Pn+Pn+ M+M+ X-X- M+M+ X-X- H+H+ OH - H+H+ Electrical Neutrality: Ion Activity Equilibrium: Picture courtesy of Yiheng Wang

15. Data Test #3 NaCl solution = 0.001M BSA solution = 0.001M Initial conductivity = 119.9 μS/cm ISE reading = 0.0009M pH = 6.9 Conductivity Temperature in μS/cm in °C 114.9 26.2 113.4 26.0 112.3 24.2 103.3 24.3 97.3 23.4 79.4 26.0 71.7 29.8 Overall change = 48.2 μS/cm Test #4 NaCl solution = 0.01M BSA solution = 0.001M Initial conductivity = 1066 μS/cm ISE reading = 0.0081M pH = 6.431 ConductivityTemperature in μS/cm in °C 804 26.7 655 25.4 446 23.3 246.9 26.7 230.9 25.1 Overall change = 835.1 μS/cm

16. Data (cont.) Test #5 NaCl solution = 0.1M BSA solution = 0.001M Initial conductivity = 10.17 mS/cm ISE reading = 0.0736M pH = 7.159 ConductivityTemperature in mS/cm in °C 5.27 23.0 3.03 28.7 1.194 27.0 Overall change = 8.976 mS/cm

17. Discussion Volume loss affecting conductivity readings Volume loss affecting conductivity readings Better accuracy with larger volumes Better accuracy with larger volumes

18. Future Objectives Improve experimental model to increase accuracy Improve experimental model to increase accuracy Measure ion binding in high concentration protein solutions Measure ion binding in high concentration protein solutions Control pH to determine its affect on ion binding Control pH to determine its affect on ion binding

19. Acknowledgements Thanks to Dr. Victor Rodgers & the B2K Group, UCR, Denise Sanders, Jill Brady, and the BRITE program. Thanks to Dr. Victor Rodgers & the B2K Group, UCR, Denise Sanders, Jill Brady, and the BRITE program. Questions? Shelby Bickford at shelby.fawnb@gmail.com

20. Goals and Questions Ion binding as a function of: Ion binding as a function of: - Ionic concentration - pH -Protein concentration Electroneutrality condition Electroneutrality condition Ability to measure each species of ion Ability to measure each species of ion

21. Donnan Equilibrium and Electroneutrality The albumin molecule is not uniformly charged within the primary structure. At neutral pH, Peters (1985) calculated a net charge of -10, -8, and 0 for domains I, II, and III for bovine serum albumin. http://www.friedli.com/research/PhD/chapter5a.htmlPeters http://www.friedli.com/research/PhD/chapter5a.html Since the BSA molecule is negatively charged, and is said to bind mostly anions, how can electroneutrality be satisfied? If cations and anions are in a 1:1 ratio, how will there ever be enough cations to balance both systems? www.nature.com/.../n6994/fig_tab/429822a_F1.html