Adriana P. AguirreDr. V. G. J. Rodgers

Department of BioengineeringUniversity of California, Riverside

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

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).

Image from Botanical Society of America

Osmotic Pressure vs. [BSA]

[BSA] (g/L)

0 100 200 300 400 500

Osm

otic

Pre

ssur

e,

(psi

)

0

20

40

60

80

100

pH = 7.4pH = 5.4pH = 4.5

Data From Yousef et al. (1998)

Δπ ≈ 15 psi

II II II I1 12 32 2 3 1

II II I1 1 12 2

N 1 N N NRT lnV N N N

pH [NaCl] M

0.15

4.5 11.595.4 10.627.4 8.81

Theoretical Protein-Ion Binding (moles NaCl / mole BSA) Theoretical Protein-Ion Binding (moles NaCl / mole BSA) (Yousef (Yousef et alet al. (1998)). (1998))

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

Free Ions

Debye Length

Hydrated Macromolecule

Free Solvent

Introduction – Protein-Ion BindingIntroduction – Protein-Ion Binding

Yousef et al. (2002)

Previous research by Scatchard et al. (1950) has shown that proteins bind several chloride ions but probably no sodium ions

Introduction – Protein-Ion Introduction – Protein-Ion Binding (cont.)Binding (cont.)

To understand the effects of Ionic Strength pH

Protein: BSA, 66 kDSalt: 0.0015 M, 0.015 M, 0.15 M NaClSemi-permeable membrane: 3.5 kD Cutoff,

CelluloseDialysis setup – solvent and protein solution

separated by a semi-permeable membrane

ResultsResults

pH [NaCl] M

0.0015

0.015 0.15 0.15Yousef et al.

(1998)

4.5 5.8 6.3 3.0 11.595.4 N/A N/A N/A 10.627.4 6.3 5.8 4.3 8.8110.0 6.6 6.3 4.6 N/A

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

Further address protein-ion binding Quantify the effects of

Protein-proton binding Protein-hydronium binding Hydrated sodium chloride

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

This research was supported by the NSF

Thanks to Dr. Rodgers, Devin W. McBride and the BRITE program