Hydrophobic Self-Association

A (macro)emulsion is formed when two immiscible Hquids, usually water and a hydrophobic organic solvent, an oil, are mechanically agitated (5) so that one Hquid forms droplets in the other one. A microemulsion, on the other hand, forms spontaneously because of the self-association of added amphiphilic molecules. During the emulsification agitation both Hquids form droplets, and with no stabilization, two emulsion layers are formed, one with oil droplets in water (o /w) and one of water in oil (w/o). However, if not stabilized the droplets separate into two phases when the agitation ceases. If an emulsifier (a stabilizing compound) is added to the two immiscible Hquids, one of them becomes continuous and the other one remains in droplet form. 

The theory of hydrophobic interaction [70-72] indicates that hydrophobic residues tend to associate with one another so as to minimize the surface area exposed to the aqueous phase and thereby to release a maximum number of structured water molecules. Therefore, the steric fit between the hydrophobic groups may be an important factor for the hydrophobic association. It is reasonable to consider that aromatic hydrophobic groups may undergo tighter hydrophobic self-association because planar aromatic rings would sterically fit with each other to favor the release of structured water. 

Hydrophobic interaction refers to the tendency of nonpolar compounds to self-associate in an aqueous environment. This self-association is driven neither by mutual attraction nor by what are sometimes incorrectly referred to as hydrophobic bonds. Self-association arises from the need to minimize energetically unfavorable interactions between nonpolar groups and water. 

Loss of the native conformation of a protein generally exposes hydrophobic amino acid residues that are normally buried on the inside of the self-associated structure and are shielded from the aqueous environment. This leads to association between the exposed hydrophobic residues of neighboring proteins (aggregation) or between these exposed residues and hydrophobic surfaces that the protein may encounter either in the manufacturing process or in the primary package. 

One inherent property of peptides that interact with membranes is that self-association or even aggregation will interfere with solubilization by organic solvents or micelles. The preparation, purification and sample preparation of extremely hydrophobic (often transmembrane) peptides is nontrivial and has been addressed by only a few papers [74—79]. 

The mechanism of reversed-phase chromatography arises from the tendency of water molecules in the aqueous-organic mobile phase to self-associate by hydrogen bonding. This ordering is perturbed by the presence of nonpolar solute molecules. As a result, solute molecules tend to be excluded from the mobile phase and are bound by the hydrophobic stationary phase. This solvophobic... 

Physical properties of the protein structure should be considered in designing strategies to achieve stable formulations because they can often yield clues about which solution environment would be appropriate for stabilization. For example, the insulin molecule is known to self-associate via a nonspecific hydrophobic mechanism66 Stabilizers tested include phenol derivatives, nonionic and ionic surfactants, polypropylene glycol, glycerol, and carbohydrates. The choice of using stabilizers that are amphiphilic in nature to minimize interactions where protein hydrophobic surfaces instigate the instability is founded upon the hydro-phobic effect.19 It has already been mentioned that hydrophobic surfaces prefer... 

Insulin Lispro was the first recombinant fast-acting insulin analogue to gain marketing approval (Table 8.3). It displays an amino acid sequence identical to native human insulin, with one alteration — an inversion of the natural proline lysine sequence found at positions 28 and 29 of the insulin jS-chain. This simple alteration significantly decreased the propensity of individual insulin molecules to self-associate when stored at therapeutic dose concentrations. The dimerization constant for Insulin Lispro is 300 times lower than that exhibited by unmodified human insulin. Structurally, this appears to occur as the change in sequence disrupts the formation of inter-chain hydrophobic interactions critical to self-association. 

Fig. 17. Fluorous patches direct the pairing of protein segments in lipid micelles. The hydrophobic peptides partition into lipid micelles, forming a-helices. Then, the superhydrophobic hexafluoroleucine residues seek each other, causing self-association into dimers and higher order aggregates. Fluorine is light, while the backbone of the a-helices is dark. From Ref. [81], with permission.

Surfactants having an appropriate hydrophobic/hydrophilic balance (sodium bis(-2-ethylhexyl)sufosuccinate, or AOT, for example) undergo concentration-dependent self association in apolar solvents to form reversed or inverted micelles (Fig. 33) [256-262]. Reversed micelles are capable of solubilizing a large number of water molecules (AOT reversed micelles in hexane are able to take up 60 water molecules per surfactant molecule, for example). Reversed-micelle-entrapped water pools are unique they differ significantly from bulk water. At relatively small water-to-surfactant ratios (w = 8-10, where w = [H20]/[Surfactant]), all of the water molecules are strongly bound to the surfactant headgroups. Substrate solubilization in the restricted water pools of reversed micelles results in altered dissociation constants [256, 257, 263-265], reactivities [256, 258, 266], and reaction products [267]. 

In an aqueous medium these wtermolecular attractive interactions make a strong contribution to biopolymer self-association and inclusion complex formation, as well as to the flocculation of biopolymer-coated colloidal particles. // //Y/molecular hydrophobic interactions commonly influence the level of folding/unfolding of macromolecules as well as their detailed conformations. 

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