muramidase and 2-2-4-trimethylpentane

The refolding kinetics of the reduced, denatured hen egg white lysozyme in sodium bis(2-ethylhexyl)sulfosuccinate (AOT)-isooctane-water reverse micelles at different water-to-surfactant molar ratios has been investigated by fluorescence spectroscopy and UV spectroscopy. The oxidative refolding of the confined lysozyme is biphasic in AOT reverse micelles. When the water-to-surfactant molar ratio (omega 0) is 12.6, the relative activity of encapsulated lysozyme after refolding for 24 h in AOT reverse micelles increases 46% compared with that in bulk water. Furthermore, aggregation of lysozyme at a higher concentration (0.2 mM) in AOT reverse micelles at omega 0 of 6.3 or 12.6 is not observed; in contrast, the oxidative refolding of lysozyme in bulk water must be at a lower protein concentration (5 microM) in order to avoid a serious aggregation of the protein. For comparison, we have also investigated the effect of AOT on lysozyme activity and found that the residual activity of lysozyme decreases with increasing the concentration of AOT from 1 to 5 mM. When AOT concentration is larger than 2 mM, lysozyme is almost completely inactivated by AOT and most of lysozyme activity is lost. Together, our data demonstrate that AOT reverse micelles with suitable water-to-surfactant molar ratios are favorable to the oxidative refolding of reduced, denatured lysozyme at a higher concentration, compared with bulk water.

Topics: Dioctyl Sulfosuccinic Acid; Hydrogen-Ion Concentration; Kinetics; Micelles; Muramidase; Octanes; Oxidation-Reduction; Protein Denaturation; Protein Folding; Protein Renaturation; Solubility; Spectrometry, Fluorescence; Surface-Active Agents; Water

Structural modifications of AOT/water/isooctane reverse micelles due to incorporation of proteins were studied at various water contents and protein concentrations, using small-angle X-ray scattering (SAXS) experiments under static conditions, rheometry analysis, and SR-SAXS experiments under induced shear flow. Two proteins, lysozyme (pI 11.1, Mw 14,300 Da) and BSA (pI 4.3, Mw 66,700 Da), were chosen as models. SAXS analysis of protein-containing reverse micelles at low water content detected minima in the average micelle size versus protein concentration curve, for both proteins, below and above their isoelectric point. This minimum was attributed to changes in the size distribution of the reverse micelles. SAXS measurements of reverse micelles at high water content have shown them to have a cylindrical form. Incorporation of lysozyme at pH 7 into the cylindrical micelles induced a shape transition to spherical micelles, which was associated with a decreased viscosity. SR-SAXS measurements under induced shear flow and dynamic conditions revealed alignment of the cylindrical micelles in the flow direction. The anisotropy parameter, a measure of the degree of the spatial order, was found to increase with increasing shear rate and to decrease with increasing lysozyme concentration.

Topics: Micelles; Muramidase; Octanes; Proteins; Scattering, Small Angle; Succinates; Surface-Active Agents; Water; X-Ray Diffraction

The reversed micellar extraction (AOT/isooctane system) using the phase transfer method was investigated in relation to the AOT concentration and the water solubilization for ribonuclease A, lysozyme and cytochrome c. The minimal AOT concentration required for 100% forward extraction was obtained for these proteins. At the minimal AOT concentration, the hydrophilic surroundings, i.e. the molar ratio of water to extracted protein in the organic phase, were independent of the protein concentration for each protein. The hydrophilic surroundings of these proteins were linearly related with Fisher's polarity ratio, p, as an index of the hydrophobicity of the protein. Using this linear relation, a procedure to estimate the sufficient AOT concentration for the protein extraction was proposed. In the cases of cytochrome c and lysozyme, the water concentration was larger than that in the protein-free system in spite of the same AOT condition. On the contrary, in the case of ribonuclease A, this large water uptake in the organic phase was not observed. These differences of water uptake were discussed in relation to the location of the protein in the AOT reversed micelles.

Topics: Animals; Cattle; Chickens; Cytochrome c Group; Eggs; Horses; Hydrogen-Ion Concentration; Isoelectric Point; Micelles; Muramidase; Myocardium; Octanes; Pancreas; Ribonuclease, Pancreatic; Water

The extraction of lysozyme, alpha-chymotrypsin, and pepsin from buffered salt solutions into reverse micelles was examined at different pH values and surfactant concentrations. The reverse micelles was formed by mixing aqueous buffer supplemented with KCl and an organic phase of isooctane(2,2,4-trimethylpentane), containing the anionic surfactant, Aerosol O. T. (dioctyl ester of sodium sulfosuccinic acid). The technique of dynamic laser scattering was used to measure the size of reverse micelles which were in equilibrium with the aqueous phase. It was found that the size of the reverse micelles decreased with increasing ionic strength but increased with increasing AOT concentration. In the process of extraction, the reverse micelles might have rearranged themselves to host the protein. The sizes of protein-filled and -unfilled reverse micelles were different, and an open equilibrium could be reached between them. Under the extraction conditions, only a small number of micelles were found to contain protein.

Topics: Chymotrypsin; Dioctyl Sulfosuccinic Acid; Hydrogen-Ion Concentration; Micelles; Molecular Structure; Muramidase; Octanes; Pepsin A; Water