Water pool, reverse micelle

To avoid this difficulty, one technique is to use reverse micelles. These materials can host a protein in a small water pool. Reverse micelles are spherical aggregates formed by dissolving amphiphiles in organic solvents. The polar head of the amphiphilic molecule is in the interior of the aggregate and the hydrophobic tail is in the organic phase. The micellar suspension is transparent, and controlled amounts of water can be added. 

Structures of W/O microemulsions are similar to water pool reverse micelles, and ionic and hydrophilic reactants, which are excluded from the bulk solvent, are highly concentrated in the small microdroplets of water [6,19,20,37], As a result, many reactions of water-soluble reactions are very rapid in these reverse micelles or W/O microemulsions. Much of the work on reactivity and acid-base equilibria has been reviewed [6,112], especially as regards effects of the size of the water pool. ... 

The water pool description of reverse micelles and O/W microemulsions is not appropriate if only small amounts of water are present. In that event the surfactants form ion pairs or small ion clusters with associated water [118]. These clusters are catalytically very effective in the decarboxylation of 6-nitrobenzisoxazole-3-carboxylate ion (3), and for solutions of cationic surfactants and hydrophobic ammonium ions rate constants of reaction in CH2CI2 decrease significantly when there is sufficient water to generate water pool reverse micelles [119,120]. Similar results were obtained for the spontaneous hydrolysis... 

The strong interactions between the water molecules also become obvious from NMR measurements by Tsujii et al..57) 13C-NMR experiments were used for determining the microviscosity of water in reversed micelles of dodecylammonium-propionate with 13C glycine cosolubilized. It was found that the apparent viscosity of the water-pool corresponds to the viscosity of a 78 % aqueous glycerol solution, obviously as a consequence of the extended network formation by strong hydrogen bonding. 

Much interest has been focused on solubilizing various amounts of water in reverse micelles. The micellar solutions can solubilize considerable amounts of water this is bound to the polar groups of the surfactant molecules by ion-dipole or dipole-dipole attraction. The properties of water solubilized by RMs are different from those of bulk water and are sensitive to the water pool parameter, Wo = [H20]/[Surfactant]. Assuming the water molecules in the oil droplets are spherical, the radius of the sphere is expressed as (Luisi et al., 1988) ... 

It is tempting to use the classical concept of pH and pKa, but several difficulties arise when applying these concepts to confined water in reverse micelles. Since the water in reverse micelles is a new solvent, the conventional determination of pH of the water pool is difficult. The micellar solubilization of oil-insoluble dyes is a well known phenomenon (Arkin and Singleterry, 1948 Fendler, 1984 Klevens, 1950 Rodgers, 1981 Ross, 1951). 

Hence, the exiplex has a sandwich structure which promotes efficient back e transfer at the water pool, and the ion yield is very small. However, a sandwich reactant pair of this sort is not formed on a micelle surface and back reaction is slower than the escape of the cation from the surface. Hie swollen micelle and microemulsion systems lead to both randomly organised ionic products and sandwich pairs, to varying extents, which are reflected in the observed yield of ions, with polar derivatives of pyrene, e.g. pyrene sulfonic acid, etc., the reactants are kept on the assembly surface where reaction occurs, giving rise to ions from a non-sandwiched type of configuration. In the reverse micellar system, these ions although they are formed, nevertheless have a short lifetime, as they cannot escape to any great distance in the small water pool. Huts, micelles are far superior to microemulsions in various aspects of... 

Reverse micelles are normally used in enzyme-catalyzed reactions. The water in the core of the micelle is called the water pool. At a constant surfactant concentration, the amount of water introduced determines the micellar size. The nature of the entrapped water in reverse micelles has been a subject of considerable debate. At low amounts of water, it is thought that most of it is bound, leading to low enzyme activity. At higher amounts, the water becomes more free with a resultant increase in enzymatic activity. 

Confinement brings novel features into the dynamics of water in reverse micelles. Markedly non-exponential deeay exists due to two different behaviors, near the surfaee and inside the pool, at least for intermediate to large reverse micelles (Wo > 4). For larger sizes, relaxation changes from the slow behavior near the surfaee to the fast relaxation at the pool [5]. 

At the present time, "interest in reversed micelles is intense for several reasons. The rates of several types of reactions in apolar solvents are strongly enhanced by certain amphiphiles, and this "micellar catalysis" has been regarded as a model for enzyme activity (. Aside from such "biomimetic" features, rate enhancement by these surfactants may be important for applications in synthetic chemistry. Lastly, the aqueous "pools" solubilized within reversed micelles may be spectrally probed to provide structural information on the otherwise elusive state of water in small clusters. 

In the past few years, a range of solvation dynamics experiments have been demonstrated for reverse micellar systems. Reverse micelles form when a polar solvent is sequestered by surfactant molecules in a continuous nonpolar solvent. The interaction of the surfactant polar headgroups with the polar solvent can result in the formation of a well-defined solvent pool. Many different kinds of surfactants have been used to form reverse micelles. However, the structure and dynamics of reverse micelles created with Aerosol-OT (AOT) have been most frequently studied. AOT reverse micelles are monodisperse, spherical water droplets [32]. The micellar size is directly related to the water volume-to-surfactant surface area ratio defined as the molar ratio of water to AOT,... 

The observation of slow, confined water motion in AOT reverse micelles is also supported by measured dielectric relaxation of the water pool. Using terahertz time-domain spectroscopy, the dielectric properties of water in the reverse micelles have been investigated by Mittleman et al. [36]. They found that both the time scale and amplitude of the relaxation was smaller than those of bulk water. They attributed these results to the reduction of long-range collective motion due to the confinement of the water in the nanometer-sized micelles. These results suggested that free water motion in the reverse micelles are not equivalent to bulk solvation dynamics. 

Investigation of water motion in AOT reverse micelles determining the solvent correlation function, C i), was first reported by Sarkar et al. [29]. They obtained time-resolved fluorescence measurements of C480 in an AOT reverse micellar solution with time resolution of > 50 ps and observed solvent relaxation rates with time constants ranging from 1.7 to 12 ns. They also attributed these dynamical changes to relaxation processes of water molecules in various environments of the water pool. In a similar study investigating the deuterium isotope effect on solvent motion in AOT reverse micelles. Das et al. [37] reported that the solvation dynamics of D2O is 1.5 times slower than H2O motion. 

The aqueous cores of reverse micelles are of particular interest because of their analogy with the water pockets in bioaggregates and the active sites of enzymes. Moreover, enzymes solubilized in reverse micelles can exhibit an enhanced catalytic efficiency. Figure B4.3.1 shows a reverse micelle of bis(2-ethylhexyl)sulfosuccinate (AOT) in heptane with three naphthalenic fluorescent probes whose excited-state pK values are much lower than the ground-state pK (see Table 4.4) 2-naphthol (NOH), sodium 2-naphthol sulfonate (NSOH), potassium 2-naphthol-6,8-disulfonate (NSOH). The spectra and the rate constants for deprotonation and back-recombination (determined by time-resolved experiments) provide information on the location of the probes and the corresponding ability of their microenvironment to accept a proton , (i) NDSOH is located around the center of the water pool, and at water contents w = [H20]/[A0T] >... 

Fig. B4.3.1. Schematic illustration of the average residence sites of the probes NOH (1), NSOH (2), NDSOH (3) in AOT reverse micelles. Length of the surfactant 11 A. Diameter of the water pool 18A at w = 3, 36A at w = 9. Largest dimension of the naphthol derivatives 9 A (adapted from Bardez et al.a ).

Figure 1 shows a reversed micelle where the bulk solvent is a hydrocarbon and the core is a water pool surrounded by surfactant. These systems possess unique features as the physical properties of the water pools only start to approach those of bulk water at high water content when the pool radii are >150 pools with radii as small as 15 can be constructed (1, 25). These systems have been used to investigate the nature of several inorganic reactions by stopped flow methods (26, 27). They have also been used to produce so-called naked ions, i.e., ions that possess a minimum of aqueous solvation (28). The system strongly promotes many reactions, a fact attributed to the unusual nature of the water in this system. 

Usually, activities of enzymes (hydrogenases included) are investigated in solutions with water as the solvent. However, enhancement of enzyme activity is sometimes described for non-aqueous or water-limiting surroundings, particular for hydrophobic (or oily) substrates. Ternary phase systems such as water-in-oil microemulsions are useful tools for investigations in this field. Microemulsions are prepared by dispersion of small amounts of water and surfactant in organic solvents. In these systems, small droplets of water (l-50nm in diameter) are surrounded by a monolayer of surfactant molecules (Fig. 9.15). The water pool inside the so-called reverse micelle represents a combination of properties of aqueous and non-aqueous environments. Enzymes entrapped inside reverse micelles depend in their catalytic activity on the size of the micelle, i.e. the water content of the system (at constant surfactant concentrations). 

Effect of salt type and concentration The ionic strength of the aqueous solution in eontaet with a reverse micelle phase affects protein partitioning in a number of ways [18,23]. The first is through modification of electrostatic interactions between the protein surface and the surfaetant head groups by modifieation of the eleetrieal double layers adjacent to both the eharged inner mieelle wall and the protein surface. The second effect is to salt out the protein from the mieelle phase because of the inereased propensity of the ionie speeies to migrate to the micelle water pool, reduee the size of the reverse mieelles, and thus displace the protein. 

Reverse micelles of CTAB in octane with hexanol as cosurfactant were reported to be able to lyse whole cells quickly and accommodate the liberated enzyme rapidly into the water pool of surfactant aggregates [50,51]. In another case a periplasmic enzyme, cytochrome c553, was extracted from the periplasmic fraction using reverse micelles [52]. The purity achieved in one separation step was very close to that achieved with extensive column chromatography. These results show that reverse micelles can be used for the extraction of intracellular proteins. 

Surfactants can aggregate in nonpolar solvents in the presence of small amounts of water with the tails oriented towards the bulk nonpolar solution and head groups interacting with water in the center (Fig. 2). The water pool formed in reverse micelles has been used as a medium to study chemical and biological reactions [22]. 

Photoinduced electron transfer from eosin and ethyl eosin to Fe(CN)g in AOT/heptane-RMs was studied and the Hfe time of the redox products in reverse micellar system was found to increase by about 300-fold compared to conventional photosystem [335]. The authors have presented a kinetic model for overall photochemical process. Kang et al. [336] reported photoinduced electron transfer from (alkoxyphenyl) triphenylporphyrines to water pool in RMs. Sarkar et al. [337] demonstrated the intramolecular excited state proton transfer and dual luminescence behavior of 3-hydroxyflavone in RMs. In combination with chemiluminescence, RMs were employed to determine gold in aqueous solutions of industrial samples containing silver alloy [338, 339]. Xie et al. [340] studied the a-naphthyl acetic acid sensitized room temperature phosphorescence of biacetyl in AOT-RMs. The intensity of phosphorescence was observed to be about 13 times higher than that seen in aqueous SDS micelles. 

Reverse micelles are small (1-2 nm in diameter), spherical surfactant aggregates huilt in an apolar solvent (usually referred to as oil), whereby the polar heads form a polar core that can contain water - the so-called water pool. The connection with autopoiesis is historically important, because it was with the collaboration with Francisco Varela that the work started (in fact it began as a theoretical paper - see Luisi and Varela, 1990). The idea was this to induce a forced micro-compartmentalization of two reagents, A and B, which could react inside the boundary (and not outside) to yield as a product the very surfactant that builds the boundary (Figure 7.13). The product S would concentrate at the membrane interface, which increases its size. Since reverse micelles are usually thermodynamically stable in only one given dimension, this increase of the size-to-volume ratio would lead to more micelles. Thus the growth and multiplication would take place from within the structure of the spherically closed unit, be governed by the component production of the micellar structure itself, and therefore (as will be seen better in... 

Figure 7.13 The first self-reproduction scheme conceived for reverse micelles, (a) A reverse micelle, (b) Two reagents, A and B, penetrate inside the water pool and react with each other inside the boundary, yielding the very surfactant S that makes the micelles. The S thus produced migrates to the boundary and induces growth and eventually multiplication of the micelle. (Adapted from Luisi and Varela, 1990).

Reverse micelles form in aprotic organic solvents, such as hydrocarbons or CCI4, and can be seen as a core containing water (the water pool) solubilized in an oily environment (for example hydrocarbons) by the hydrophobic tails. Figure 9.9 also shows the structure of AOT (from aerosol octyl), which is the most popular surfactant for reverse micelles. A typical reverse micellar system appears as a clear... 

Figure 9.10 Some structural details and dynamic properties of reverse micelles 50 irtM AOT/isooctane, Wo = 11.1 (= 10 p lHoOperml), 25°C 3.2% AOT (w/w), 1.4% H2O (w/w) mean water pool radius 20 A, mean hydrohynamic radius 32 A concentration of micelles 400 (xM, monomer AOT concentration 0.6-0.9 mM aggregation number 125 total interfacial area 14 m mC (Adapted from Fletcher and Robinson, 1981, and Harada and Schelly, 1982.)...

Figure 9.11 A case of selective compartmentation in reverse micelles, permitting the synthesis of a peptide by the reverse protease action. The product C, produced in the water pool, is expelled into the outside hydrocarbon environment due to its insolubility in water. (Adapted from Barbaric and Luisi, 1981.)...

---