This document summarizes a study on the effect of monovalent salt anions on the osmotic pressure of bovine serum albumin (BSA). Osmotic pressure data was obtained for BSA solutions containing 0.15 M NaCl, NaF, or NaI at pH 7.4. Using a free-solvent model, ion binding and hydration values were calculated from the experimental osmotic pressure data. The results showed that ion binding values differed from some literature values and were not entirely consistent with the Hofmeister series. Further osmotic pressure experiments are planned to gather additional data up to BSA saturation limits to improve the model sensitivity and understanding of how ion binding influences osmotic pressure.

1. Protein (MW kDa)
Soln Properties:
Salt Conc., pH
Hydration Ion Binding
BSA (66.5)
0.15 M NaCl, 4.5
0.15 M NaCl, 5.4
0.15 M NaCl, 7.4
1.113 ± 0.0063
1.137 ± 0.0059
1.177 ± 0.0050
11.59[2]
10.62[2]
8.81[2]
Effect of Monovalent Salt Anions on the Osmotic Pressure of
Bovine Serum Albumin
Larry Chang, Danielle N. Ornelas, Noriko Uka Ozaki-Felt, Victor G. J. Rodgers
Department of Bioengineering, University of California, Riverside
References
1. Yousef MA, Datta R, Rodgers VGJ. AIChE J. 2002; 48:913-917.
2. Yousef MA, Datta R, Rodgers VGJ. J Colloid Interface Sci. 1998; 207:273-282.
3. Yousef MA, Datta R, Rodgers VGJ. J Colloid Interface Sci. 2001; 243:321-325.
4. Reboiras MD, Pfister H, Pauly H. Biophysical Chemistry. 1986; 24:249-257.
5. Scatchard G, Scheinberg IH, Armstrong SH. J Am Chem Soc. 1950; 72:535-540.
6. Zhang Y and Cremer PS. Current Opinion Chem Bio. 2006; 10:658-663.
› The presence of anions alters osmotic pressure and the overall
hydration of the system.
› The FSM was able to calculate ion binding and hydration with
reasonable error.
› Ion binding and hydration is not monotonically consistent with the
Hofmeister series.
› Results show that our experimentally determined ion binding values
were significantly different than those calculated by Reboiras et al.
and Scatchard et al.
› Further osmotic pressure data is needed for high BSA concentrations
up to saturation limit in NaI. Sensitivity of the model increases as
BSA concentration increases .
› Further osmotic pressure data will be gathered for BSA in 0.15 M
NaF, NaCl, NaI, and NaSCN at pH 4.5.
T h e B 2 K G r o u p │ B I O T R A N S P O R T & B I O R E A C T I O N K I N E T I C S
For additional information, please contact Dr. Victor G. J. Rodgers at
vrodgers@engr.ucr.edu.
CO3
2- SO4
2- S2O3
2- H2PO4
- F- Cl- Br- NO3
- I- ClO4
- SCN-
Discussion and Future Work
Aggregation Solubilization
dp dn12( )
Objective
The free-solvent model predicts the non-linear protein osmotic pressure
of concentrated protein solutions at moderate ionic strength. Here, we
investigate how anions affect the osmotic pressure of bovine serum
albumin (BSA) in the presence of 0.15 M NaCI, NaF, NaI and NaSCN at
pH 7.4. Osmotic pressure data are obtained up to saturation. Using non-
linear least squares regression, we are able to predict the model
parameters of ion binding and hydration values. The results will allow
further understanding of how osmotic pressure is coupled to ion binding
and hydration relative to the Hofmeister series.
Free-Solvent Model (FSM)
pH Dependence of Ion Binding Observed from
Osmotic Pressure
Hofmeister Series
Literature F-, Cl-, I-, and SCN- Ion Binding to Albumin
Ion Binding and Hydration of 0.15 M Salt Solutions
Osmotic Pressure of Bovine Serum Albumin (BSA)
The FSM was developed to describe non-ideal behavior of proteins at
high concentrations. FSM is an ideal model that places emphasis on the
hydrated macromolecule. The FSM considers the non-attractive
interactions between macromolecules and the remaining free solvent.
There are two biologically significant parameters that are independently
determined when predicting protein osmotic pressure; protein hydration,
v1j, and salt ion binding, v3j.The mole fraction of diffusible species, ,
is also critical when determining the prediction of osmotic pressure.
x1
II
x1
I
p =
RT
V1
ln
N1
I
Ni
II
i=2
n
å - nij Nj
II
j=2
p+1
åi=1,i¹2®p+1
n
å( )
NI
N1
II
- n1j Nj
II
j=2
p+1
å( )
æ
è
ç
ç
ç
ö
ø
÷
÷
÷
Studies previously conducted by
Yousef et al. showed that while
the hydration values are relatively
consistent, the salt ion binding
values were sensitive among
varying pH. However, salt ion and
albumin interactions are limited
due to the number of charged
surface residues.
The Hofmeister series is a classification of ions in order of their ability to
salt out or salt in proteins. The early members of the series decrease the
solubility of nonpolar molecules, strengthening the hydrophobic
interactions. The later salts increase the solubility of nonpolar molecules,
while weakening the hydrophobic effect.
Reboiras et al. utilized an empirical model to examine the binding of
potassium salts to BSA at high protein concentrations. Scatchard et al.
developed an empirical model for providing expected ion binding of
sodium chloride to human serum albumin (HSA).






BSAg
OHg 2






BSAmol
Saltmol
1: water
2p: proteins
(p+1)n: salt
I: solvent chamber
II: protein chamber
36 psi
Ion
Soln Properties:
Salt Conc. [M]
Ion Binding
Soln. Properties:
Salt Conc. [M]
Ion Binding
F- 0.1 3[4] 0.3 -2[4]
Cl- 0.1 6[4], 8[5] 0.3 3[4], 12[5]
I- 0.1 11[4] 0.3 16[4]






BSAmol
Saltmol






BSAmol
Saltmol
Theoretical
Saturation Limit
Soln Properties:
Salt Conc., pH
Ion Binding Hydration
601.32
0.15 M NaF, 7.4
0.15 M NaF, 7.4
6.18 ± 0.645
3[4]
0.927 ± 0.037
0.696 ± 0.014
544.96 0.15 M NaCl, 7.4 6.79 ± 0.658 1.099 ± 0.033
724.11
0.15 M NaI, 7.4
0.15 M NaI, 7.4
-0.29 ± 1.156
11[4]
0.645 ± 0.054
1.175 ± 0.010
Soln Properties:
Salt Conc., pH
Second Virial
Coefficient, β2 × 10-7
Ionic Strength
Ionic Strength
Ratio, α
0.15 M NaF, 7.4 2.4 0.100 0.667
0.15 M NaCl, 7.4 4.8 0.093 0.621
0.15 M NaI, 7.4 2.6 -0.007 -0.045
*At 400 g/L protein concentration.
L × mol
g2
æ
è
ç
ö
ø
÷
mol salt
vol H2O
æ
è
ç
ö
ø
÷
mol salt
vol total species
æ
è
ç
ö
ø
÷
g L( )






BSAmol
Saltmol






BSAg
OHg 2
2
2