Quantifying the Protein-Ion Binding Phenomenon

1. Quantifying the Protein-Ion Binding Phenomenon Adriana P. Aguirre Dr. V. G. J. Rodgers Department of Bioengineering University of California, Riverside

2. Introduction – Macromolecular Crowding • Crowded protein solutions are found in nature, and are solutions containing 50 – 450 mg/mL of proteins • In crowded protein solutions, the movement of protons cause a flux of bulk water across a membrane, subsequently causing an increase in osmotic pressure • The mechanism of Venus Flytrap closure is thought to be driven by osmotic pressure Image from Botanical Society of America Minton, A.L., “The Influence of Macromolecular Crowding and Macromolecular Confinement on Biochemical Reactions in Physiological Media”, The Journal of Biological Chemistry, 276(14) 10577 (2001).

3. Introduction – Osmotic Pressure vs. BSA Δπ ≈ 15 psi Data From Yousef et al. (1998)

4. Introduction – Free Solvent Model Theoretical Protein-Ion Binding (moles NaCl / mole BSA) (Yousef et al. (1998))

5. Free Solvent Debye Length Hydrated Macromolecule Free Ions Introduction – Protein-Ion Binding • Protein-ion binding is the interaction of proteins with small molecules. • Both water and ions bind to proteins to create a hydrated protein, which can be viewed as a single molecule Image adapted from Dr. Victor G. J. Rodgers Yousef et al. (2002)

6. Introduction – Protein-Ion Binding (cont.) Previous research by Scatchard et al. (1950) has shown that proteins bind several chloride ions but probably no sodium ions

7. Objectives To understand the effects of • Ionic Strength • pH

8. Procedure Protein: BSA, 66 kD Salt: 0.0015 M, 0.015 M, 0.15 M NaCl Semi-permeable membrane: 3.5 kD Cutoff, Cellulose Dialysis setup – solvent and protein solution separated by a semi-permeable membrane

9. Procedure – Experiment in Action

10. Results

11. Salt Binding to BSA (moles salt/mole BSA)

12. Conclusion • As the ionic strength increases, the protein-ion binding decreases • This trend does not follow Scatchard’s work • As the pH increases, the protein-ion binding increases

13. Future Studies • Further address protein-ion binding • Quantify the effects of • Protein-proton binding • Protein-hydronium binding • Hydrated sodium chloride

14. References 1. Minton, A.L., “The Influence of Macromolecular Crowding and Macromolecular Confinement on Biochemical Reactions in Physiological Media”, The Journal of Biological Chemistry, 276(14) 10577 (2001). 2. Yousef, M. A., Datta, R., and Rodgers, V. G. J., “Understanding Non-Idealities of the Osmotic Pressure of Concentrated Bovine Serum Albumin”, Journal of Colloid and Interface Science, 207(2), 273-282 (1998). 3. Yousef, M. A., Datta, R., and Rodgers, V. G. J., “Confirmation of Free-Solvent Model Assumptions in Predicting the Osmotic Pressure of Concentrated Globular Proteins”, Journal of Colloid and Interface Science, 243, 321-325 (2001). 4. George Scatchard, I. Herbert Scheinberg and S. Howard Armstrong, JR. (1950) Physical Chemistry of Protein Solutions. IV. The Combination of Human Serum Albumin with Chloride Ion. 5. http://www.botany.org/carnivorous_plants/venus_flytrap.php

15. Acknowledgements • This research was supported by the NSF • Thanks to Dr. Rodgers, Devin W. McBride and the BRITE program

16. Questions ?