Ionic strength enzymatic activity

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). 

The enzymes differ significantly in their optimal conditions for enzymatic activity. These include pH, ionic strength and temperature. The variety of conditions enabled us to verify if there could be limits to the approach. 

Another important parameter is the acidity of the system. Since the relation between enzyme activity and pH often shows an optimum, buffers are regularly used in enzymatic derivatization to control the pH. Use of buffers, however, can cause increase of the ionic strength of the system, thus changing the tertiary structure of the enzyme and either making it more accessible for the substrate or blocking its active sites. Therefore, optimization of the chromatographic system is recommended for each specific system. 

The rate of an enzymatic reaction is affected by a number of environmental factors, such as solvent, ionic strength, temperature, pH, and presence of inhibitor/activator. Some of these effects are described below. 

Bivalent ion (concentration 10-s M) Enzymatic activity determined at ionic strength" ... 

Researchers, primarily from Moscow State University, Russia, have also formed noncovalent aggregates between enzymes and polymers [palmitoylated poly (sucrose acrylate)], polyelectrolytes (polybrene), and block copolymers. Reported enzymatic activities were high. For the former, activity decreased as the molecular weight and degree of palmitoylation increased. Chymotrypsin-polyelectrolyte and -block copolymer complexes were active in aqueous-polar organic co-solvent mixtures when the ionic strength was increased. " ... 

The rate of an enzymatic reaction depends on a number of factors, including the temperature, pH, ionic strength, and so forth. The rate of the reaction will increase as the temperature is increased, up to a point. Above a certain temperature, the activity of the enzyme is decreased because, being a protein, it becomes denatured, that is, the tertiary structure of the enzyme is destroyed as hydrogen bonds are broken. The steric nature of an enzyme is critical in its catalytic mechanism. Most animal enzymes become denatured at temperatures above about 40°C. 

DSC, reported that SCN, another chaotropic anion, stabilizes BSA in the same conditions. It is known that SCN binds strongly to BSA [191]. We observed [195] that Ca2+ and Mg2+ decrease QD and Tm of ribulose 1,5-diphosphate carboxylase (Rubisco) to a limited extent but in a definite and steady way as their concentration increases over the ionic strength range 0-0.3 mol/L Na+ and NH+4 had an opposite effect. In the case of Rubisco, specificity of the effect of Ca2+ and Mg2 + is supported by the fact that these cations are known to bind to the protein and to affect its enzymatic activity by inducing structural changes. Specific effects of Na+ and Ca2+ are also observed by DSC on the heat stability of a-lactalbumin [190] they result in a linear increase of QD with the cation/protein molar ratio up to 1, followed by a plateau (Fig. 7). 

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