Conformation, reverse micelles

As mentioned above, water structure in reversed micelles deviates considerably from the structure in the bulk-phase. Therefore, the hydration shell of macromolecules entrapped in reversed micellar systems should be changed and thus also their conformation. According to the results of several authors this is indeed the case. 

The conformation of bovine myelin basic protein (MBP) in AOT/isooctane/water reversed micellar systems was studied by Waks et al. 67). This MBP is an extrinsic water soluble protein which attains an extended conformation in aqueous solution 68 but is more density packed at the membrane surface. The solubilization of MBP in the AOT reversed micelles depends on the water/AOT-ratio w0 68). The maximum of solubilization was observed at a w0-value as low as 5.56. The same value was obtained for another major protein component of myelin, the Folch-Pi proteolipid 69). According to fluorescence emission spectra of MBP, accessibility of the single tryptophane residue seems to be decreased in AOT reversed micelles. From CD-spectra one can conclude that there is a higher conformational rigidity in reversed micelles and a more ordered aqueous environment. 

The conformational dynamics of chain segments near the head groups is more restricted than that of those far from the micellar core [8]. Moreover, to avoid the presence of energetically unfavorable void space in the micellar aggregate and as a consequence of the intermolecular interactions, surfactant molecules tend to assume some preferential conformations and a staggered position with respect to the micellar core [9]. A schematic representation of a reversed micelle is shown in Figure 1. 

As a result of the micellar environment, enzymes and proteins acquire novel conformational and/or dynamic properties, which has led to an interesting research perspective from both the biophysical and the biotechnological points of view [173-175], From the comparison of some properties of catalase and horseradish peroxidase solubilized in wa-ter/AOT/n-heptane microemulsions with those in an aqueous solution of AOT it was ascertained that the secondary structure of catalase significantly changes in the presence of an aqueous micellar solution of AOT, whereas in AOT/n-heptane reverse micelles it does not change. On the other hand, AOT has no effect on horseradish peroxidase in aqueous solution, whereas slight changes in the secondary structure of horseradish peroxidase in AOT/n-heptane reverse micelles occur [176],... 

For many solubilized enzymes the greatest catalytic activity and/or changes in conformation are found at R < 12, namely, when the competition for the water in the system between surfactant head groups and biopolymers is strong. This emphasizes the importance of the hydration water surrounding the biopolymer on its reactivity and conformation [13], It has been reported that enzymes incorporated in the aqueous polar core of the reversed micelles are protected against denaturation and that the distribution of some proteins, such as chymotrypsine, ribonuclease, and cytochrome c, is well described by a Poisson distribution. The protein state and reactivity were found markedly different from those observed in bulk aqueous solution [178,179],... 

In order to be exploitable for extraction and purification of proteins/enzymes, RMs should exhibit two characteristic features. First, they should be capable of solubilizing proteins selectively. This protein uptake is referred to as forward extraction. Second, they should be able to release these proteins into aqueous phase so that a quantitative recovery of the purified protein can be obtained, which is referred to as back extraction. A schematic representation of protein solubilization in RMs from aqueous phase is shown in Fig. 2. In a number of recent publications, extraction and purification of proteins (both forward and back extraction) has been demonstrated using various reverse micellar systems [44,46-48]. In Table 2, exclusively various enzymes/proteins that are extracted using RMs as well as the stability and conformational studies of various enzymes in RMs are summarized. The studies revealed that the extraction process is generally controlled by various factors such as concentration and type of surfactant, pH and ionic strength of the aqueous phase, concentration and type of CO-surfactants, salts, charge of the protein, temperature, water content, size and shape of reverse micelles, etc. By manipulating these parameters selective sepa-... 

Barbaric, S. and Luisi, P. L. (1981). Micellar solubilization of biopolymers in organic solvents. 5. Activity and conformation of a-chymorypsin in isooctane-AOT reverse micelles. J. Am. Chem. Soc., 103,4239 4. 

Bile salts are natural and chiral anionic surfactants which form helical micelles of reversed micelle conformation. The first report on enantiomer separation by MEKC using bile salts was the enantioseparation of... 

It was noted that activity and conformation change with the amount of water inside micelles, pointing out the importance of the aqueous environment, easily adjustable as a function of the water content, which is impossible when studies are performed in aqueous solutions. Furthermore, because many properties (9) of the water core resemble those of water present at interfaces in biological systems, reversed micelles provide an excellent system for studying the interactions between polypeptides and interfacial water (10-11) or more generally their conformation when solubilized in micelles (12-15), depending on where the biopolymer is located inside the micelle and what its conformation is. 

Many surfactants are known to form reversed micelles in apolar media and have already provided a suitable environment for elucidating catalytic activity or conformation properties of some proteins in non aqueous media. But to conduct an extraction two conditions must be fulfilled reversed micelles must exist in the organic phase in equilibrium with an excess aqueous phase and the performance of the extraction must be significant. The nature of both surfactant and solvent, the composition... 

These assumptions are confirmed by experiments with the spin-labeled active center of a-chymotrypsin [41] (Fig. 6). As seen from the data in Fig. 6, the enzyme entrapped in reverse micelles is located in the medium with decreased polarity. In similar polar media of water-organic mixtures, enzyme structures are normally disrupted, and protein denatura-tion and loss of catalytic enzymic activity occur. Yet, in systems of reverse micelles a principally different picture is observed. In optimal enzyme activity conditions the protein becomes tightly fixed by the micellar matrix and its conformational mobility is minimized. 

From the above results, it is assumed that the interaction of the hydrophobic and cationic moieties of HK with a negatively charged layer of AOT results in its insertion into the AOT layer, which leads to the suppression of conformational change. On the other hand, the HK conformation in the HTAC reversed micelles may be more easily changed because of the location of HK relatively far from the interface of the water pool due to the electrostatic repulsion and the larger size of the micelles compared with that of the AOT reversed micelles. Consequently, the high electrostatic interface of AOT and HTAC is not favorable for the appearance of HK catalytic activity. 

The structure of Ci2Eg reversed micelles is greatly different from those of AOT and HTAC reversed micelles, as described in Section II. It is assumed that HK exists in the hydrated EO mantle, binds easily with the substrate, and changes its conformation to reveal catalytic activity. The HK activity in the Ci2Eg reversed micelles is of interest... 

For an ionic surfactant such as AOT, increasing salinity leads to a 2-3-2 transition. The AOT-brine-propane system conforms to this classical behavior in several ways. For example, increases in the salinity of the aqueous phase increase the proportion of AOT in the propane phase. At high pressures, the size of the reverse micelles (as reflected by JVo) decreases as salinity increases. The pressure at which the 2-3 transition occurs decreases as salinity increases, an indication of increasing surfactant affinity for the propane phase. One interesting aspect of the AOT-brine-propane system is that the amount of NaCI required to effect phase changes appears to be higher than is the case in conventional liquid solvents. For propane at 310 bar and 37°C, about 1.1 wt% salt is required to drive AOT into the propane phase. For the heptane-brine-AOT system at the same concentration, only 0.5 wt% salt is required to achieve the same effect [36]. This result suggests that propane is a weaker solvent for AOT than heptane. 

The anomalous activity characteristics have been attributed to conformational changes of the solubilized enzyme [49], but more recent spectroscopic studies seem to indicate that this is not the main cause. Solubilization of an enzyme into microemulsion droplets does not normally lead to major conformational alterations, as indicated, e.g., by fluorescence and phosphorescence spectral investigations [28,50]. The situation is complex, however, and it has been shown by circular dichroism (CD) measurements that the influence of the oil/water interface on enzyme conformation may vary even between enzymes belonging to the same class [51]. In the case of human pancreatic lipase, the conformation of the polypeptide chain is hardly altered after the enzyme is transferred from a bulk aqueous solution to the microenvironment of reverse micelles. Conversely, the CD spectra of the lipases from... 

There have been many studies devoted to characterization of these practically important systems. Reverse emulsion droplets have been used as chemical micro-reactors to produce nanosize inorganic and polymer particles with special properties that are not found in the bulk form (38-42). These microemulsion systems have also been a topic of research for biological systems and the AOT head groups have been found to influence the conformation of proteins and increase enzyme activity (43-6). The unique environment created in the small water pools of swollen reverse micelles allows for increased chemical reactivity. The increase in surface area with decreased in size of the droplets also can significantly increase reactivity by allowing greater contact of immiscible reactants. 

A very different kind of surfactant molecule obtains if the carboxylate head group of an alkanoic acid is replaced by one that is both hydrophobic and lipophobic, like a perfluoroalkyl group. Such diblock molecules, F(CF2) (CH2) H(FmHn), have been shown to form normal and reverse micelles in perfluoroalkanes and alkanes, respectively [68,69]. On the bases of the well-known antipathy between hydrocarbons and perfluorocarbons, the disparity between cross-sectional areas and volumes of CH (18.5 and 10.22 A ) [70, 71] and CF (28.3 A and 16.04 A ) [70, 71] groups, and the tendency of long perfluoroalkyl chains to adopt helical conformations, it is expected and found that organized assemblies of FmH/i molecules exhibit some strange properties. For instance, the alkyl portions of many of these molecules melt before their perfluoroalkyl portions [72], and the allowed motions resemble closely those of -alkanes in their rotator phases [73, 74]. 

Bile salts are natural and chiral anionic surfactants which form helical micelles of reversed micelle conformation. The first report on enantiomer separation by MEKC using bile salts was the enantioseparation of dansylated DL-amino acids (Dns-o,L-AAs) and, since then, numerous papers have been available. Nonconjugated bile salts, such as sodium cholate (SC) and sodium deoxycholate (SDC), can be used at pH > 5, whereas taurine-conjugated forms, such as sodium taurocholate (STC) and sodium taurodeox-ycholate (STDC), can be used under more acidic conditions (i.e., pH > 3). Several enantiomers, such as diltiazem hydrochloride and related compounds, carboline derivatives, trimetoquinol and related compounds, binaphthyl derivatives, Dhs-dl-AAs, mephenytoin and its metabolites, and 3-hydroxy-l,4-benzodiazepins have been successfully separated by MEKC with bile salts. In general, STDC is considered as the the most effective chiral selector among the bile salts used in MEKC. 

The enzymes embedded within the reverse micelles retain full enzymic activity it is noteworthy, however, that many of them are found to undergo an increase in conformational rigidity , i.e., in the percentage of secondary structure (e.g. helix content), and that the effect is more marked the lower the water content of the micellar system. The special microenvironment in which biopolymer molecules are enclosed might therefore exert a marked effect on their conformation, stability and activity. 

A knowledge of water pool pH(pHwp) is extremely useful when one attempts to rationalize the effects of the water pool microenvironments on the conformations of a reaction participation by biopolymers in reverse micelles. 

One such generalization seems to be the following that the biopolymers in reverse micelles undergo an increase in conformational rigidity (defined in a thermodynamic sense [96]), or more precisely, an increase in the percent of secondary structure (e. g. helix content). The effect is more marked the smaller the water content in the micellar system in other words, the effect is more marked when the micelle size is smaller and the water of the water pool is more anomalous with respect to bulk water. 

Let us see some examples of this effect. Figure 3 shows the case of lysozyme in reverse micelles with 0.3 and 3% water vis-a-vis bulk water. As already reported [89], the increase of helix content is marked, and also in the near uv-region, where the spectrum is mostly sensitive to the aromatic side chains, there are large alterations. Since marked alterations are not visible in uv-absorption spectroscopy, the changes in CD can be safely ascribed to conformational changes. 

What are enzymes in micelles good for From the previous pages, it is apparent that these systems may elicit curiosity in the chemist. Changes in conformation and activity of enzymes with respect to aqueous solution, the anomalous structure of water in the water pool and its relation to enzyme activity, the conceptual problems of the local pH and the local concentration, and, generally speaking, the picture of the reverse micelle as a peculiar microreactor where enzymatic and nucleic add reactions can take place in novel ways - all of this presents a fascinating research enterprise. 

You are right. In fact, preliminary experiments show that lysozyme in reverse micelles is conformationally much more stable in the presence of NAG3 (see B. Stemmann, M. Jackie, P. L. Luisi, Biopolymers, in press - added in the proofs). 

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