Aqueous media protein solubilization

Water-in-oil microemulsions (w/o-MEs), also known as reverse micelles, provide what appears to be a very unique and well-suited medium for solubilizing proteins, amino acids, and other biological molecules in a nonpolar medium. The medium consists of small aqueous-polar nanodroplets dispersed in an apolar bulk phase by surfactants (Fig. 1). Moreover, the droplet size is on the same order of magnitude as the encapsulated enzyme molecules. Typically, the medium is quite dynamic, with droplets spontaneously coalescing, exchanging materials, and reforming on the order of microseconds. Such small droplets yield a large amount of interfacial area. For many surfactants, the size of the dispersed aqueous nanodroplets is directly proportional to the water-surfactant mole ratio, also known as w. Several reviews have been written which provide more detailed discussion of the physical properties of microemulsions [1-3]. 

The role of catalysis in membrane assembly is emphasized again by the above model since the N-terminal sequence of the nascent polypeptide chain of a spanning protein is released by proteolysis as soon as it reaches the cytosol. The N-terminal polypeptide chain extension may help the chain penetrate the hydrophobic bilayer and solubilize the resulting hydrophobic N-terminal part of the chain in the aqueous medium of the cytoplasm. However, the role of the protease-catalyzed hydrolysis of the polypeptide chain in membrane assembly is minimized in the membrane trigger hypothesis (99). According to this model, the essential role of the leader sequence would be to modify, in association with the lipid bilayer, the folding pathway of the protein in such a way that the polypeptide chain could span the membrane. 

Gel electrophoresis is widely used in the routine analysis and separation of many well-known biopolymers such as proteins or nucleic acids. Little has been reported concerning the use of this methodology for the analysis of synthetic polymers, undoubtedly since in many cases these polymers are not soluble in aqueous solution - a medium normally used for electrophoresis. Even for those water-soluble synthetic polymers, the broad molecular weight dispersities usually associated with traditional polymers generally preclude the use of electrophoretic methods. Dendrimers, however, especially those constructed using semi-controlled or controlled structure synthesis (Chapters 8 and 9), possess narrow molecular weight distribution and those that are sufficiently water solubile, usually are ideal analytes for electrophoretic methods. More specifically, poly(amidoamine) (PAMAM) and related dendrimers have been proven amendable to gel electrophoresis, as will be discussed in this chapter. 

In the neutral red (cell viability) and total protein (cell proliferation) assays, cells are treated with various concentrations of a test substance in petri or multiwell dishes after a period of exposure, the substance is washed out of the medium. (An analytical reagent is added in the case of protein measurements.) Neutral red is a supravital dye, which accumulates in the lysosomes of viable, uninjured cells, and it can be washed out of cells, which have been damaged. In the protein test, Kenacid blue is added and reacts with cellular protein. Controlled cells are dark blue killed cells are lighter colored. The IC50 (the concentration which inhibits by 50%) is determined the test can be rapidly performed with automation. However, materials must be solubilized into the aqueous cell media for analysis. For many test materials this will require large dilutions which eliminate properties of the materials which cause irritation. 

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