Protein extraction studies mechanisms

Methods. Early investigations relevant to technological analyses of the freezing operations involved determining the amount of protein extractable in salt solutions, such as 5% NaCl or 0.6M KC1. Proteins extracted in this manner were defined as native or undenatured. Then a question arose concerning the mechanism by which denaturation occurs. Unfortunately, denatured proteins are difficult to study because of their insolubility thus information about the state of proteins in fish had to be gained from the soluble protein fraction. 

Recent studies have shown that HIAR is also effective for physically fixed materials, un-embedded and plastic-embedded specimens, and immunoelec-tron microscopy. Heating has also been shown to be useful for the extraction of proteins and nucleic acids from FFPE materials. The elucidation of the mechanisms described above should support the usefulness of such applications and lead to the establishment of standardized protocols for immunohis-tochemical staining. 

Several kinetic parameters can be measured on different experimental systems to account for the interaction of a compound with CYPs. For example when studying the metabolic stability of a compound, it could be measured in a recombinant CYP system, in human liver microsomes, in hepatocytes and so on. Each system increases in biological complexity. Although in the recombinant CYP system only the cytochrome under consideration is studied, in the case of the human liver microsomes, there is a pool of enzyme present that includes several CYPs, and finally in the hepatocyte cell system, metabolizing enzymes play an important role in the metabolic compound stability. In addition, transport systems are also present that could involve recirculation or other transport phenomena. The more complex the experimental system, the more difficult it is to extract information on the protein/ligand interaction, albeit it is closer to the in vivo real situation and therefore to the mechanism that is actually working in the body. 

The mechanism of separation of biological molecules such as proteins and amino acids, and the parameters that affect the extraction distribution coefficient and the kinetics of extraction have been studied more extensively than the extraction of inorganic solutes. This is mainly due to the variety of size and structure of these molecules and, furthermore, to the fact that their characteristics may be adversely affected by their contact with solvents and surfactants. 

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