Preferential exclusion

Properly folded native proteins tend to aggregate less than when unfolded. Solution additives that are known to stabilize the native proteins in solution may inhibit aggregation and enhance solubility. A diverse range of chemical additives are known to stabilize proteins in solution. These include salts, polyols, amino acids, and various polymers. Timasheff and colleagues have provided an extensive examination of the effects of solvent additives on protein stability [105]. The unifying mechanism for protein stabilization by these cosolvents is related to their preferential exclusion from the protein surface. With the cosolvent preferentially excluded, the protein surface is... 

Differential hydration of proteins has been little exploited as a selectivity factor in ion exchange, but it is simple to evaluate and can produce useful results. This technique relies on the preferential exclusion of certain solutes from protein surfaces to produce an exclusionary effect and favor their interaction with the column. Protein hydration is generally proportional to protein size and solubility. Among proteins of similar size, this predicts that retention will increase with protein solubility. Among proteins of similar solubility, retention increases with protein size.16... 

The PEG could stabilize proteins by two different temperature-dependent mechanisms. At lower temperatures, it is preferentially excluded from the protein surface but has been shown to interact with the unfolded form of the protein at higher temperatures, given its amphipathic nature (57). Thus, at lower temperatures, it may protect proteins via the mechanism of preferential exclusion, but at higher temperatures possibly by reducing the number of productive collisions between unfolded molecules. PEG is also a cryoprotectant and has been employed in Recombinate, a lyophilized formulation of recombinant Antihemophilic Factor, which utilizes PEG 3350 at a concentration of 1.5mg/mL. The low-molecular weight liquid PEGs (PEG 300-600) can be contaminated with peroxides and cause protein oxidation. If used, the peroxide content in the raw material must be minimized and controlled throughout its shelf life. The same holds true for polysorbates (discussed below). 

PHYSICAL CHEMISTRY OF PROTEIN-WATER-SOLUTE INTERACTIONS PREFERENTIAL EXCLUSION AND SOLVOPHOBIA... 

A second mechanism of preferential exclusion is based on the steric properties of the protein and cosolvent. The three-dimensional structure of the cosolvent may hinder interactions with the protein surface. High-molecular-weight cosolvents like polyethylene glycols (PEGs) and other proteins may be preferentially excluded through this steric hindrance mechanism. 

The thermodynamic description of protein stabilization, which involves the concept of preferential exclusion of stabilizing cosolvents, and the chemical description of osmophobicity of peptide linkages to stabilizing cosolvents represent two sides of a coin. The symmetry in these descriptions can perhaps best be appreciated by viewing osmophobicity as arising from the creation by a stabilizing cosolvent like TMAO of an aqueous phase whose structure is not favorable for hydration of the pep-... 

The exclusion of stabilizing solutes from the water adjacent to proteins may also be reduced as temperature is increased, although this effect of temperature on water s properties is not well understood. The preferential exclusion mechanism for protein stabilization, like hydrophobi-cally based stabilization, thus could be temperature dependent and cease to make a strong contribution to protein stability at high temperatures. 

Cryo- and Lyoprotectants and Bulking Agents Various mechanisms are proposed to explain why excipients serve as cryo- or lyoprotectants. The most widely accepted mechanism to explain the action of cryoprotection is the preferential exclusion mechanism [177]. Excipients that will stabilize proteins against the effects of freezing do so by not associating with the surface of the protein. Such excipients actually increase the surface tension of water and induce preferential hydration of the protein. Examples of solutes that serve as cryoprotectants by this mechanism include amino acids, polyols, sugars, and polyethylene glycol. 

Several authors reported measurements of the preferential binding parameter in the system water (l)/protein (2)/PEG (3) [10—14]. It was found that for various proteins, various PEGs molecular weights, and various PEG concentrations, the protein is preferentially hydrated and the PEG is excluded from the vicinity of the protein molecule. The prevalent viewpoint which explains such a behavior is based on the steric exclusion mechanism suggested by Kauzmann and cited in Ref. [15]. According to this mechanism [12,14], the deficit of PEG and the excess of water (in comparison with the bulk concentrations) are located in the shell (volume of exclusion) between the protein surface and a sphere of radius R (see Fig. 1) [12,14]. However, Lee and Lee [10,11] suggested that the preferential exclusion of the PEG from the protein surface also involves the protein hydrophobicity and charge. 

The excess (or deficit) numbers of molecules of water and PEG (A i2 and A 23) around a protein molecule were calculated using Eq. (16) and are listed in Table 1. The results demonstrate that in all cases there is preferential exclusion of PEG from the surface of the protein, conclusion in agreement with previous observations [10-14]. There is only one exception (water/ lysozyme/PEG 200, at a concentration of 10 g PEG/100 ml solution). However, this is probably caused by the inaccuracy in the experimental value of F y (according to the authors of Ref [12], for this composition, 7 2 3 =0.66 1.32 [mol/mol]). 

Sugars and polyols at low concentrations have been shown to increase the solubility of certain proteins, but at high concentrations decrease solubility due to preferential exclusion from the protein surface and competition with water (Arakawa and Timasheff, 1985 Middaugh and Volkin, 1992 Jenkins, 1998). 

There are two main mechanisms of solvent-induced stabilization of proteins (i) strengthening of the protein-stabilizing forces or (ii) destabilization of the denatured state (18). The most tenable and widely accepted mechanism of protein stabilization in aqueous solution is the preferential interaction of proteins. Preferential interaction indicates that a protein prefers to interact with either water or the excipient. The two conventionally applied terms are preferential hydration, which means that a protein prefers to interact with water, or preferential exclusion, which means that for example the excipient is... 

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