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