Proteins association with small molecules

Quantitation of small molecules with enzymatic methods provides insight into the ccMicentration and activity of the proteins associated with those molecules. A great variety of these enzymatic assays have been carried out in microfluidic devices. Another function of enzymatic assays is in kinetics measurements of properties of enzymes such as the Michaelis-Menten constant (the concentration of substrate when the reactimi rate is half the maximum rate) and the turnover number (the number of moles of substrate that are converted to product per catalytic site per unit time) are vital to understanding the mechanics of the proteome and are used to characterize the effects of known drugs and discover new ones. 

The precise mechanism(s) by which oligos enter cells is not fully understood. Most are charged molecules, sometimes displaying a molecular mass of up to 10-12 kDa. Receptor-mediated endocytosis appears to be the most common mechanism by which charged oligos, such as phosphorothioates, enter most cells. One putative phosphorothioate receptor appears to consist of an 80 kDa surface protein, associated with a smaller 34 kDa membrane protein. However, this in itself seems to be an inefficient process, with only a small proportion of the administered drug eventually being transferred across the plasma membrane. 

The separation of small peptides is fairly well understood however, it appears that no single approach can be applied to the separation of large peptides or proteins. To a great extent, this is because of the wide diversity and complexity associated with these molecules. As a result, different techniques must be employed for different protein separation problems. 

Redox proteins are relatively small molecules. In biological systems they are membrane associated, mobile (soluble) or associated with other proteins. Their molecular structure ensures specific interactions with other proteins or enzymes. In a simplified way this situation is mimicked when electrodes are chemically modified to substitute one of the reaction partners of biological redox pairs. The major classes of soluble redox active proteins are heme proteins, ferredoxins, flavoproteins and copper proteins (Table 2.1). In most cases they do not catalyze specific chemical reactions themselves, but function as biological (natural) electron carriers to or between enzymes catalyzing specific transformations. Also some proteins which are naturally not involved in redox processes but carry redox active sites (e.g., hemoglobin and myoglobin) show reversible electron exchange at proper functionalized electrodes. 

Once a protein structure has been solved, the study of the association of small molecules with the protein may be accomplished relatively easily by means of difference Fourier syntheses. The method has been widely applied in the study of binding of inhibitors and pseudo-substrates to a large number of proteins and has provided the means by which active and allosteric sites may be located. It is assumed that the small ligand does not change the unit cell or perturb the protein substantially, and that the protein phases are approximately equal to those for the protein and ligand. Small changes in conformation can be distinguished as in conventional difference syntheses. The coefficients used are... 

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