Temperature control, enzyme activity

Enzyme Assays. An enzyme assay determines the amount of enzyme present in sample. However, enzymes are usually not measured on a stoichiometric basis. Enzyme activity is usually determined from a rate assay and expressed in activity units. As mentioned above, a change in temperature, pH, and/or substrate concentration affects the reaction velocity. These parameters must therefore be carefully controlled in order to achieve reproducible results. 

Available methods provide measurements of enzyme activity rather than of enzyme concentration. In order that the measured activity be proportional to enzyme concentration, the reaction conditions which include pH, temperature, initial substrate concentration, sample and total volume and reaction time must be held constant and be carefully controlled. 

Reagents. The measurement of enzyme activities requires rigid control of the analytical conditions, including accurate measurement of reagent and sample volumes, and careful control of temperature, pH and reagent stability. 

Approximation refers to the bringing together of the substrate molecules and reactive functionalities of the enzyme active site into the required proximity and orientation for rapid reaction. Consider the reaction of two molecules, A and B, to form a covalent product A-B. For this reaction to occur in solution, the two molecules would need to encounter each other through diffusion-controlled collisions. The rate of collision is dependent on the temperature of the solution and molar concentrations of reactants. The physiological conditions that support human life, however, do not allow for significant variations in temperature or molarity of substrates. For a collision to lead to bond formation, the two molecules would need to encounter one another in a precise orientation to effect the molecular orbitial distortions necessary for transition state attainment. The chemical reaction would also require... 

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 ability of some organisms to control the pH and temperature of their cells and tissues represents a major biological development. Homeothermic animals (e.g. mammals) maintain a constant temperature of about 37 °C as this corresponds to the temperature of optimum activity of most enzymes. Poikilothermic or so-called cold-blooded animals (e.g. reptiles) have to sun themselves for sometime every morning in order to raise their body temperature in order to optimize enzyme activity within their cells. 

Basal enzyme levels for control fish in the Venezuelan crude experiment (Table V) were higher than the control values for experiments with pure hydrocarbons (Table IV). Increase in basal activity was correlated with an increase in water temperature and the initiation of feeding. Enzyme activity was consistently low in cunners during the winter, and appears to rise in late spring to a summer peak (24). 

Most ultrasonic experiments are carried out in temperature controlled systems to ensure that isothermal conditions are maintained. Even a small general increase in microbial temperature can influence both the active and passive transport systems of the cell membrane/wall and this in turn may lead to an increased uptake of compounds. If the temperature is not controlled then sonication could result in a large temperature increase which will lead to the denaturation (deactivation) of enzymes, proteins and other cellular components present within the microorganism [7]. 

In summary, we have shown for the first time that catalysts such as enzymes may be immobilized within LCST hydrogels. These gels may be warmed above their LCST s, significantly reducing the enzyme activity of the gel. We have also shown that this phenomenon is reversible. We propose that such gels may be used to control reaction rates and temperatures by a feedback mechanism. 

The conditions used in an enzyme assay depend on what is to be accomplished by the assay. There are two primary applications of an enzyme assay procedure. First, it may be used to measure the concentration of active enzyme in a preparation. In this circumstance, the measured rate of the enzyme-catalyzed reaction must be proportional to the concentration of enzyme stated in more kinetic terms, there must be a linear relationship between initial rate and enzyme concentration (the reaction is first order in enzyme concentration). To achieve this, certain conditions must be met (1) the concentrations of substrate(s), cofactors, and other requirements must be in excess (2) the reaction mixture must not contain inhibitors of the enzyme and (3) all environmental factors such as pH, temperature, and ionic strength should be controlled. Under these conditions, a plot of enzyme activity (p-rnole product formed/minute) vs. enzyme concentration is a straight line and can be used to estimate the concentration of active enzyme in solution. 

Cells regulate their metabolic activities by controlling rates of enzyme synthesis and degradation and by adjusting the activities of specific enzymes. Enzyme activities vary in response to changes in pH, temperature, and the concentrations of substrates or products, but also can be controlled by covalent modifications of the protein or by interactions with activators or inhibitors. 

There are many factors that influence the outcome of enzymatic reactions in carbon dioxide. These include enzyme activity, enzyme stability, temperature, pH, pressure, diffusional limitations of a two-phase heterogeneous mixture, solubility of enzyme and/or substrates, water content of the reaction system, and flow rate of carbon dioxide (continuous and semibatch reactions). It is important to understand the aspects that control and limit biocatalysis in carbon dioxide if one wants to improve upon the process. This chapter serves as a brief introduction to enzyme chemistry in carbon dioxide. The advantages and disadvantages of running reactions in this medium, as well as the factors that influence reactions, are all presented. Many of the reactions studied in this area are summarized in a manner that is easy to read and referenced in Table 6.1. 

Any process, industrial or laboratory-based, presents a series of important variables that represent its state. In the case of cell culture, there are the variables related to the environment to which the cells are exposed, such as temperature, pH, dissolved oxygen, nutrients in the culture medium, and metabolite concentrations, as well as those related to the cell itself, such as concentration, average size, or the profile of intracellular enzyme activities. In order to make meaningful decisions during a process it is desirable to have at hand the greatest amount of information available about these variables. At this point, it is important to define some concepts relative to process monitoring and control. 

The effect of temperature satisfies the Arrhenius relationship where the applicable range is relatively small because of low and high temperature effects. The effect of extreme pH values is related to the nature of enzymatic proteins as polyvalent acids and bases, with acid and basic groups (hydrophilic) concentrated on the outside of the protein. Finally, mechanical forces such as surface tension and shear can affect enzyme activity by disturbing the shape of the enzyme molecules. Since the shape of the active site of the enzyme is constructed to correspond to the shape of the substrate, small alteration in the structure can severely affect enzyme activity. Reactor s stirrer speed, flowrate, and foaming must be controlled to maintain the productivity of the enzyme. Consequently, during experimental investigations of the kinetics enzyme catalyzed reactions, temperature, shear, and pH are carefully controlled the last by use of buffered solutions. 

Environmental factors such as pH, ionic strength, and temperature affect enzyme activity. They must be controlled when making in vitro measurements of enzyme activity and heeded in vivo when abnormal conditions such as acidosis, alkalosis, and fevers may exist. 

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