Denaturation interaction between factors

Dilute solutions are used experimentally because one is interested in studying the intramolecular interactions responsible for the stabilization of the structure rather than any interactions between different molecules. Other factors, such as aggregation and reduced solubility of the protein in the denatured state, also make this the concentration region of choice. j Other scientists sometimes refer to it as a second-law enthalpy. 

The studies also revealed some factors that affect the stability of a protein at the electrode/electrolyte interface. On an electrode surface, the native conformation of a protein may be retained or distorted, depending on the extent of the interactions between them. Because in most of the water-soluble proteins the hydrophilic side chains are usually located on the exterior, irreversible adsorption and denaturation of proteins are expected to be considerably less on a hydrophilic electrode surface at which intervening water molecules are more tenaciously bound. Therefore, adsorption at both modified gold and edge-... 

It is well known that a particular conformation of a biopolymer maintains its stability only in aqueous solutions. Addition of, say, 20% alcohol causes a conformational change which eventually leads to the process of denaturation. The latter is a very complex process and Involves the combination of many factors such as hydrogen bonding, ionic interaction, and van der Waals interaction between the various residues of the polymer. It has been conjectured that the tendency of the nonpolar groups (such as methyl or ethyl groups attached to the amino acids) to avoid the aqueous environment is one of the major reasons for the stabilization of the native conformation of the biopolymer. This is shown schematically in the first process depicted in Fig. 8.1. Here, we stress an extreme example where the polymer is folded in such a way that the side-chain nonpolar groups are completely removed from the aqueous medium and transferred to the interior of the polymer, where they are exposed to an environment similar to that of a typical nonpolar solvent. 

Enzymatic reactions are influenced by a variety of solution conditions that must be well controlled in HTS assays. Buffer components, pH, ionic strength, solvent polarity, viscosity, and temperature can all influence the initial velocity and the interactions of enzymes with substrate and inhibitor molecules. Space does not permit a comprehensive discussion of these factors, but a more detailed presentation can be found in the text by Copeland (2000). Here we simply make the recommendation that all of these solution conditions be optimized in the course of assay development. It is worth noting that there can be differences in optimal conditions for enzyme stability and enzyme activity. For example, the initial velocity may be greatest at 37°C and pH 5.0, but one may find that the enzyme denatures during the course of the assay time under these conditions. In situations like this one must experimentally determine the best compromise between reaction rate and protein stability. Again, a more detailed discussion of this issue, and methods for diagnosing enzyme denaturation during reaction can be found in Copeland (2000). 

Up to 4 kbar, protein is not denatured. However, slight structural changes can be observed, inducing flavin release from its binding site. Free flavin in solution has almost no interactions with the protein, and so energy transfer between the flavin and the amino acids of its binding site decreases, which induces an increase in the fluorescence intensity of the co-factor. 

Adsorption/absorption of biomolecules primarily results in depletion of necessary factors provided by the medium or secreted by the cells. It is an equilibrium-based phenomenon resulting in partitioning of the biomolecules between the PDMS material and the aqueous phase. Consequently, in a fluidic environment, new biomolecules (in the case of those provided by the medium) continuously arrive and are adsorbed/absorbed while a portion of the previously adsorbed/absorbed ones are detached from the PDMS material. As long as there is a sufficient supply of biomolecules in the medium, cell growth is not hampered. However, small molecules and proteins that are secreted by the cells and necessary for control of cellular functions (autocrine and paracrine factors) can potentially be removed to an extent strongly competing with the cellular capacity to excrete them. Secondly, adsorption of proteins, even if they were supplied by the medium and only transiently adsorbed on the hydrophobic surface of PDMS, causes an even more severe problem. The hydrophobic interaction of proteins with PDMS results in their denaturation due to the exposure of the hydrophobic core of the protein. 

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