Reverse micelles as hosts for proteins and small molecules.

Structure, dynamics and transport properties of microemulsions

The present state of the field of biopolymers solubilized in apolar solvents via reverse micelles is reviewed. First, an extensive discussion of the physical and chemical properties of reverse micelles is presented. Particular attention is devoted to the nature of water in the water pools of reverse micelles; to the structure and shape of the micellar aggregates; and to the dynamic properties of the reverse micelles. In the second part of the paper, the mechanism of solubilization of proteins and nucleic acids in hydrocarbon reverse micelles is discussed. Spectroscopic data, mostly circular dichroism and fluorescence, are reviewed in order to clarify the conformational changes which the biopolymers undergo upon their uptake into the micellar environment and determine the location of the biopolymers inside the reverse micelles. Data from neutron scattering, light scattering, ultracentrifugation, and electron microscopy of the protein-containing micelles are reviewed and discussed with the aim of illustrating the structure of the micellar aggregates containing the biopolymer as guest molecules. The activity of enzymes and nucleic acids is discussed, with emphasis on the influence upon the chemical reactivity brought about by the micellar parameters. Finally, a brief review of the applications and potentialities of biopolymer-containing reverse micelles is presented.

Solubilization of enzymes and nucleic acids in hydrocarbon micellar solutions.

Enzymes Hosted in Reverse Micelles in Hydrocarbon Solution

In the past twenty five years, microemulsions have been the focus of extensive research worldwide due to their importance in a variety of technological applications. These applications include enhanced oil recovery, combustion, cosmetics, pharmaceuticals, agriculture, metal cutting, lubrication, food, enzymatic catalysis, organic and bio-organic reactions, chemical synthesis of nanoparticles etc. The basic purpose of this review is intended to provide the most important features of microemulsions and their applications. Emphasis is placed on recent works, however, the reference list is by no means complete. In order to limit the size of the review, a more or less arbitary selection has been made with respect to examples chosen from literature. The review is started with a brief introduction and focussed with definition, structure, type, formation characteristics, stability, phase behavior and the effect of additives, pressure, temperature on the phase behavior of microemulsion. In addition, the p...

Microemulsions: an overview

The quaternary ammonium salt methyl-trioctylammonium chloride enables the transfer of α-chymotrypsin, trypsin, pepsin and glucagone from water to cyclohexane. Reversed micelles, whose polar core solubilizes both protein and water, are probably formed in the apolar phase. The influence of various parameters on the phase transfer (concentration, pH, solvent, temperature, etc.) has been investigated.

Absorption, fluorescence and circular dichroism studies of the biopolymers in the cyclohexane system have been carried out. For trypsin and chymotrypsin, the CD. signal in the 200 nm region is very similar in water and in cyclohexane, which suggests that the polypeptide folding is not substantially different in the two phases. The fluorescence quantum yield is always much larger in the cyclohexane phase than in water. The longer wavelength region of the UV. absorption spectrum is slightly red-shifted relative to water, and a band at 225 nm, probably arising from the aromatic chromophore, is apparent in the organic phase. Reasons for these spectral perturbations are discussed. The enzymes transferred from water into cyclohexane phases can be continuously retransferred into a second water phase. The possible relevance of this ‘double transfer’ as a model for the vectorial transport of biopolymers or a separation technique is discussed.

Micellar Solubilization of Proteins in Aprotic Solvents and their Spectroscopic Characterisation

Three proteins (horse liver alcohol dehydrogenase, ribonuclease, lysozyme) were solubilized in hydrocarbon with the help of reverse micelles formed by aqueous di(2-ethyl-hexyl) sodium sulfosuccinate (AOT). Sedimentation and diffusion coefficients of the micellar aggregates were measured with an analytical ultracentrifuge. Partial specific volumes were also evaluated from density measurements. The molecular weight of the protein-containing reverse micelles (Mt) could thus be determined for each protein system at various w0 values (w0 - [H2O]/[AOT]). For horse liver alcohol dehydrogenase at w0 = 46.4, for example,Mt is ca. 2,670,000 Daltons; for lysozyme at wo = 22.5,Mt is ca. 323,000 Daltons and increases by increasing w0. On the basis of these experimentally determined molecular weights, a structural model for the protein-containing reverse micelle is proposed. The model is based upon the assumption that the protein is confined in the water pool of a spherical micelle, and that the inner core volume is the sum of the protein volume and the volume of all water molecules present in a micelle. It is possible then to calculate the micellar structure at each w0 value. For example, in the case of ribonuclease at w0 = 20, the inner core radius is ca. 37.5 A, and a layer of water of ca. 22 A separates the protein surface from the surfactant layer. The possible implications of this model for the reactivity of enzymes solubilized in hydrocarbons by reverse micelles are discussed.

Micellar solubilization of biopolymers in hydrocarbon solvents. I. a structural model for protein-containing reverse micelles

This work reports on the interaction of the fluorescent nicotinamide 1,N6-ethenoadenine dinucleotide (epsilonNAD+) with horse liver alcohol dehydrogenase, octopine dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase from different sources (yeast, lobster muscle, and rabbit muscle). The coenzyme fluorescence is enhanced by a factor of 10-13 in all systems investigated. It is shown that this enhancement cannot be due to changes in the polarity of the environment upon binding, and that it must be rather ascribed to structural properties of the bound coenzyme. Although dynamic factors could also be important for inducing changes in the quantum yield of epsilonNAD+ fluorescence, the close similarity of the fluorescence enhancement factor in all cases investigated indicates that the conformation of bound coenzyme is rather invariant in the different enzyme systems and overwhelmingly shifted toward an open form. Dissociation constants for epsilonNAD+-dehydrogenases complexes can be determined by monitoring the coenzyme fluorescence enhancement or the protein fluorescence quenching. In the case of yeast glyceraldehyde-3-phosphate dehydrogenase at pH 7.0 and t = 20 degrees the binding plots obtained by the two methods are coincident, and show no cooperativity. The affinity of epsilonNAD+ is generally lower than that of NAD+, although epsilonNAD+ maintains most of the binding characteristics of NAD+. For example, it forms a tight complex with horse liver alcohol dehydrogenase and pyrazole, and with octopine dehydrogenase saturated by L-arginine and pyruvate. One major difference in the binding behavior of NAD+ and epsilonNAD+ seems to be present in the muscle glyceraldehyde-3-phosphate dehydrogenase. In fact, no difference was found for epsilon NAD+ between the affinities of the third and fourth binding sites. The results and implications of this work are compared with those obtained recently by other authors.

Relationship between fluorescence and conformation of epsilonNAD+ bound to dehydrogenases.

Corrected fluorescence properties of yeast alcohol dehydrogenase and its coenzyme complexes have been investigated as a function of temperature. Dissociation constants have been obtained for binary and ternary complexes of NAD and NADH by following the enhancement of NADH fluorescence or the quenching of the protein fluorescence. It is found that the presence of pyrazole increases the affinity of NAD to the enzyme approximately 100-fold. The formation of the ternary enzyme - NAD - pyrazole complex is accompanied by a large change in the ultraviolet absorption properties, with a new band in the 290-nm region. Significant optical changes also accompany the formation of the ternary enzyme-NADH-acetamide complex. The possible origin for the quenching of the protein fluorescence upon coenzyme binding is discussed, and it is suggested that a coenzyme-induced conformational change can cause it. Thermodynamic parameters associated with NAD and NADH binding have been evaluated on the basis of the change of the dissociation constants with temperature. Optical and thermodynamic properties of binary and ternary complexes of yeast alcohol dehydrogenase are compared with the analogous properties of horse liver alcohol dehydrogenase.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1976.tb10517.x

Francis J. Bonner

Papers

Micellar Solubilization of Proteins in Aprotic Solvents and their Spectroscopic Characterisation

Micellar solubilization of biopolymers in hydrocarbon solvents. I. a structural model for protein-containing reverse micelles

Relationship between fluorescence and conformation of epsilonNAD+ bound to dehydrogenases.

Spectroscopic Investigation of Binary and Ternary Coenzyme Complexes of Yeast Alcohol Dehydrogenase