1. Ananda Mishra, Cyrus Buckman, Daniel Chris Gomes, and Laurel M. Pegram
Department of Chemistry, Earlham College
Quantifying Interactions of Salts with Biologically-Relevant Surfaces
Inorganic salts and small non-electrolyte solutes exert a wide range of effects on protein and other
biomolecular processes. These effects are due to the competing molecular interactions between water and the
functional groups on salts or osmolytes and those on the biomolecule. A complete thermodynamic description
of these interactions is necessary for understanding processes that take place in cells, where salt and
cosolute concentrations are significant and variable. In this research, we used vapor pressure osmometry to
quantify the interactions between protein model compounds (amides and amino acids) and a series of
Hofmeister salts. Model compounds, including malonamide, glycine, diglycine, and N,N-dimethylmalonamide
were dissolved with and without salts (including protein denaturant GuHCl) at varying molal concentrations.
The difference between the measured osmolalities for the three-component solutions and corresponding two-
component solutions provide a quantity that can be used to interpret the preferential interactions in terms of
salt partitioning between the bulk solution and the hydration layer of the model compound.
Abstract
In 1888, Franz Hofmeister, an early protein scientist
discovered a series of cations and anions that affected
the solubility/precipitation of proteins in remarkably
consistent order.
Subsequently, an analogous nonelectrolyte series has
been discovered, and all series have been found
applicable to thermodynamic effects on a wide variety of
processes occurring in aqueous solution, e.g. surface
tension, solubility, and protein folding.
The thermodynamic origin of these effects is the
competition between the salt ions and water for the
surface of interest. The salt ions and nonelectrolyte
solutes that prefer to stay hydrated in the bulk are
excluded from the surface; this exclusion leads to burial
of surface. Conversely, those ions and solutes that
interact with the surface of interest strongly, relative to
their interactions with water, are accumulated at the
surface, which leads to exposure.
This is illustrated below for protein unfolding.
Introduction
Materials and Methods
Results and Conclusions
 The osmometrically obtained ΔOsm values are
plotted as a function of the molal concentration
product (Figs. 3 and 4); the slope is a chemical
potential derivative that is analogous to a protein
unfolding m-value and can be analyzed via the
solute partitioning model.
(+) slope: net unfavorable interaction
(-) slope: net favorable interaction
 GuHCl interacts more favorably than KCl with the
two amide model compounds. KCl has a net
unfavorable interaction with dmma.
 These results support earlier proposals that K+
ions are strongly excluded from hydrocarbon
groups and GuH+ ions are accumulated at both
hydrocarbon and amide surfaces.
 All salts investigated interact most favorably with
diglycine, due to the charged N and C termini.
 Determination of ΔOsm involves a subtraction of
three small osmolality values. KCl bracketing is
necessary to reduce the scatter caused by
instrumental drift at higher osmolalities.
References
Pegram, L. M. and Record, M. T. (2008). Thermodynamic
origin of Hofmeister ion effects. JPCB, 112, 9428-9436.
Capp, M. W.; Pegram, L. M.; Saecker, R. M.; Kratz, M.;
Riccardi, D.; Wendorff, T.; Cannon, J. G.; and Record, M. T.
(2009). Interactions of the osmolyte glycine betaine with
molecular surfaces in water: Thermodynamics, structural
interpretation, and prediction of m-values. Biochemistry, 48,
10372-10379.
Paterova, J.; Rembert, K. B.; Heyda, J.; Kurra, Y.; Okur, H.
I.; Liu, W. R.; Hilty, C.; Cremer, P. S.; and Jungwirth, P.
(2013). Reversal of the Hofmeister series: Specific ion
effects on peptides. JPCB, 117, 8150-8158.
Acknowledgements
We gratefully acknowledge the Earlham College
Collaborative Research Fund and the Gerald
Bakker Collaborative Research Endowment Fund
for summer support and the Caldwell Scientific
Equipment Fund for the instrument and supplies. We
also thank Dr. Demian Riccardi for his support and
encouragement.
Fig 2. Determination of DOsm: Osmolality of two-component and three-
component solutions plotted against molal GuHCl concentration.
Fig 1. Graph of deviations in osmolality values based on KCl bracketing
Vapor pressure osmometer
http://www.elitechgroup.com/
Future work
 Surface area calculations will be done to
decompose the model compounds into course-
grained surface area types (e.g. aliphatic C, amide
O, amide N, etc.).
 Interactions of salts with additional amide model
compounds will also need to be quantified so that
a wide range of surface area compositions are
represented in the data set.
 Net interactions can then be dissected into both
cation/anion and functional group contributions.
5
Solubilizing Precipitating
6
accumulation
surface area exposure
exclusion
surface area burial
7
urea
GuH
+
SCN
-
ClO4
-
malonamide
dimethylmalonamide
• Two-component solutions were prepared using
protein backbone model compounds as solute and
water as the solvent.
• Malonamide, dimethylmalonamide, glycine, and
diglycine were the model compounds selected.
• Three-component solutions contained water, model
compound, and a Hofmeister salt. The molal
concentrations of the model compounds were kept
constant while the molality of the added salt was
varied.
Results
Fig 3: Experimental data quantifying the interactions of GuHCl or KCl
with malonamide or dimethylmalonamide
Fig 4: Experimental data quantifying the interactions of KCl, GuH2SO4,
or GuHCl with the dipeptide glycylglycine.
• The osmolalities (a measure of nonideality) of the prepared solutions were
measured using VPO.
• Using the available literature data for osmolality as a function of KCl concentration,
we adopted a system of bracketing each solution with two KCl solutions of known
osmolality.
To gain more information about the exact nature of the
interaction of Hofmeister ions with the amide groups on
the protein backbone, we have undertaken this
thermodynamic study. Osmometric data quantifying
Hofmeister salt - model compound interactions can be
used, along with surface area calculations to decompose
the total interaction (whether unfavorable or favorable)
into interactions of the salt with component functional
groups.
glycine