Proteins separation from surfactants

The most significant problem with the utilization of surfactant media in different separation schemes (particularly those at the preparative or process scales) concerns the recovery of the analyte from the surfactant media and subsequent recovery of the surfactant for re-use. Attempts to use extraction schemes with conventional organic solvents typically results in troublesome emulsion formation during the recovery steps. There are, however, several means available by which analytes can be recovered free of surfactant. These include the following (1) Several quick, gentle methods for the recovery of some analytes (usually proteins) from surfactant media (i.e. micellar NaLS, Triton X-100, CHAPS, deoxycholate, Brij-35) via use of column chromatography have been developed (509-515). Most of the stationary phase materials for this approach are available commercially (510,513). 

We typically do not use protease inhibitors. The combination of the lysis buffer with its reducing ability, the chaotropic effects of the urea and the surfactant, and the cold temperature seems to inactivate proteolytic activity. We also do not perform any steps requiring room temperature or protein activity (such as the DNAse-RNAse treatment found in some protocols). Furthermore, the presence of the inhibitors may sometimes interfere with the fluorescent labeling. The sample cups on the lEF gel have about 100-pl maximum capacity. However, if necessary, more volume can be handled by ordering more sample cup holder bars separately from the dry strip kit and used to spread one sample between several cups. Because IFF is a focusing technique, the sample does not necessarily have to be applied in exactly the same spot. The dye synthesis is detailed elsewhere. The dyes are not commercially available as of the time of this writing. 

In order to better understand the pathophysiology associated with these various forms of pulmonary edema, a review of the morphology associated with the capillary-alveolar—intestinal interlace is useful (Fig. 6). From this review it can be seen that fluid in pulmonary capillaries is separated from the alveolar interstitial tissue by the capillary endothelial cells and the capillary basement membrane (commonly called the endothelial barrier). The alveolar surface is separated from the interstitial space by the alveolar-airway barrier, which consists of the alveolar basement membrane, alveolar epithelium, and a layer of pulmonary surfactant within the alveolus. As described previously, the alveolar interstitial tissue is made up of connective tissue (elastin and collagen), fibronectin, and mucopolysaccharides. The interstitial space also contains the pulmonary lympatic system, which functions to drain proteins, large particulate matter, and excess fluid away from the tissue space and to return them to the blood. 

The zein test developed by Gotte [6] is based on the solubilization by surfactants of a maize protein which is normally insoluble in aqueous solution, unless denaturated. The protein is incubated with the surfactant solution for 1 h, at a constant temperature and under slight shaking at the end of incubation, the soluble fraction is separated from the insoluble one by centrifugation and filtration. Solubilized zein is assayed. The more irritating the surfactant, the more zein will be denaturated and solubilized. 

The numerous separations reported in the literature include surfactants, inorganic ions, enzymes, other proteins, other organics, biological cells, and various other particles and substances. The scale of the systems ranges from the simple Grits test for the presence of surfactants in water, which has been shown to operate by virtue of transient foam fractionation [Lemlich, J. Colloid Interface Sci., 37, 497 (1971)], to the natural adsubble processes that occur on a grand scale in the ocean [Wallace and Duce, Deep Sea Res., 25, 827 (1978)]. For further information see the reviews cited earlier. 

Various methods have been used to examine the composition of proteins adsorbed to SAMs. Overall adsorption patterns can be examined with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [50, 76, 77]. Absorbed proteins are eluted from the surface with surfactant (SDS), and then separated by electrophoresis. The proteins of interest are examined by western blotting [50, 76, 77]. Protein-specific antibodies can be used to detect proteins of... 

Interest in the nature of interactions between shortchain organic surfactants and large molecular weight macromolecules and ions with hydroxyapatite extends to several fields. In the area of carles prevention and control, surfactant adsorption plays an important role in the Initial states of plaque formation (1-5) and in the adhesion of tooth restorative materials ( ). Interaction of hydroxyapatite with polypeptides in human urine is important in human biology as hydroxyapatite has been found as a major or minor component in a majority of kidney stones ( 7). Hydroxyapatite is used in column chromatography as a material for separating proteins (8-9). The flotation separation of apatite from... 

Cloud point extraction from biological and clinical samples. The most frequent use of CPE is for the separation and purification of biological analytes, principally proteins. In this way, the cloud point technique has been used as an effective tool to isolate and purify proteins when combined with chromatographic separations. Most of the applications deal with the separation of hydrophobic from hydrophilic proteins, with the hydrophobic proteins having more affinity for the surfactant-rich phase, and the hydrophilic proteins remaining in the dilute aqueous phase. The separation of biomaterials and clinical analytes by CPE has been described [105,106,113]. 

Effect of surfactant type and concentration An increase in surfactant concentration results in an increase in the number of micelles rather than any substantial change in size, and this enhances the capacity of the reverse micelle phase to solubilize proteins. Woll and Hatton [24] observed increasing protein solubilization in the reverse micelle phase with increasing surfactant concentration. In contrast, Jarudilokkul et al. [25] found that at low minimal concentrations (6-20 mmol dm AOT), reverse mieelles eould be highly seleetive in separating very similar proteins from... 

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. 

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