Protein Salting Out Observed at an Air−Water Interface

Abstract

b S Supporting Information P rotein crystallization is typically realized by the addition of a salt or an organic solvent to a supersaturated protein solution to decrease the protein solubility. "Salting out" is a method for separating proteins, in which the hydrated salt ions reduce the number of water molecules available for interaction with the proteins. Protein solubility is a macroscopic property resulting from various molecular interactions, including proteinÀprotein, proteinÀion, ionÀwater, and waterÀprotein interactions, and depends on temperature, pH, ion species, and concentration. At low salt concentrations, a "salting-in" region exists, where favorable interactions occur between the protein surface charges and the salt ions in solution to increase protein solubility, which is sometimes associated with protein unfolding. At higher salt concentrations, the repulsive electrostatic interactions are screened, resulting in protein stabilization, and proteinÀprotein interactions such as hydrophobic interactions and dispersion forces can drive aggregation and precipitation. 1À4 The effect of ion species on the solubility of proteins is classified by the well-known Hofmeister series, defined by the concentration of a particular salt needed to precipitate proteins from whole egg white. 5,6 The series are usually given in terms of the ability of the ions to stabilize the structure of proteins. Although Hofmeister ion effects on protein stability are widely reported in protein research, the mechanism is not entirely clear to date. Most previous research on Hofmeister ion effects on proteinÀprotein interactions has been performed in bulk liquids by varying ion species and concentration. 4,7À11The surface tension, that is, the surface free energy per area, can be regarded as the work in bringing a molecule from the interior of a liquid to the surface and is strongly affected by molecular forces and configurations. 12 When salt ions dissociate in water, the magnitude of the surface tension increment indicates the energy required to separate the ions from the water molecules. The molar surface tension increment of the salt has been known to follow the rank order of the Hofmeister series. 13À15 Several recent experiments and computer simulations 16 looking at interfaces have revealed that the propensity for salt anions to adsorb at an airÀwater interface follows an inverse Hofmeister series, that is, strongly hydrated anions effective in precipitating proteins do not adsorb at the airÀwater interface and vice versa. 17 These results suggest that studying the surface adsorption of proteins in a salt solution could provide valuable insight into the salting out of proteins generally.Proteins adsorb at airÀwater interfaces as surfactants. The effect of salt on protein adsorption under equilibrium conditions has been studied by surface pressure measurements, surfacespecific spectroscopies, and neutron reflectometry. 18À22 The amount of adsorption and the thickness of the interfacial layer are strongly dependent on the ...

Enzymes immobilized in Langmuir-Blodgett films: Why determining the surface properties in Langmuir monolayer is important?

“…As they are amphiphilic, the surface excess of proteins lead to the formation of Gibbs monolayers as reported in pioneering works (Gaines 1966). The amount of proteins at the interface can be increased using high salt concentrations at the aqueous subphase (Yano et al 2011), but care with possible denaturation may be taken into account. Usually, the pre-formation of a stable Langmuir monolayer at the air-water interface (formed, for instance, by lipids) may also help avoid denaturation of proteins adsorbing from the aqueous subphase since the amphiphilic nature of lipids may help conserve the structure of the polypeptide moiety of the protein.…”

Electrochemical behaviour of myoglobin at an array of microscopic liquid–liquid interfaces

“…Interestingly, the results presented here show that as the ionic strength increases (Figure 3(a-c)), the peaks attributed to protein adsorption/desorption become sharper and better defined as well as producing greater currents. A possible reason for the increase in the peak currents is the salting out effect [45]. This effect is used to precipitate proteins in solutions by adjusting the salt concentration.…”

Direct encapsulation of proteins into calcium silicate microparticles by water/oil/water interfacial reaction method and their responsive release behaviors

“…However, when the concentration of sodium chloride was very high (5 M), the protein release decreased adversely. As this 5 M solution of sodium chloride was nearly saturated (the molar concentration of the saturated sodium chloride solution is approximately 5.39 M), it is thought that this reverse behavior of lysozyme release might be due to the salting-out effect of proteins by sodium chloride [42,43]. The release profiles of lysozyme in potassium chloride solution with varied concentrations were also examined as shown in Fig.…”