Therefore, to compare the results of different assays, the volume of the organic INK 128 in vitro solvent added to the assay mixture must always be kept constant, even if the concentration of the weakly soluble substrate is reduced. The temperature dependence of the activity of enzymes resembles in some respect the pH dependence: increasing with rising temperature, passing a maximum, followed by a decrease. Therefore this behaviour is frequently described as temperature optimum, although an optimum temperature for the enzyme activity does not necessarily exist at all. Indeed,

two counter-acting processes are responsible for this behaviour ( Figure 5). The velocity of any chemical reaction increases with temperature, according to an empirical rule two to three times every 10 °C. This holds also for enzyme reactions and only boiling of water limits this progression. On the other hand the three-dimensional structure of enzymes is thermo-sensitive and becomes destabilized at high temperature causing denaturation. This process opposes the acceleration of the reaction velocity and is responsible for its decline at high temperature. The progression of denaturation depends both on the actual temperature buy LDK378 and

on time, the higher the temperature, the faster denaturation. Therefore, no fixed temperature can be given for the maximum enzyme activity; rather it depends on the pre-treatment of the enzyme. If the enzyme is immediately tested at a moderate denaturation temperature, its activity will be considerably higher than if it is kept at the same temperature for a longer time before starting the assay. Such a situation can easily arise if a certain time is needed to prepare and start the assay, while the enzyme is already present in the thermostatted assay mixture. During this time denaturation already proceeds

and since such preparation times are not always equal, the loss of the enzyme activity will also vary ( Figure 5). For assay temperatures specified in the assay protocols usually such facts are taken into account, but with special only applications, e.g. enzymes that have not yet been investigated, it should be ensured that the assay temperature is within the stability range. Some enzymes (e.g. alcohol dehydrogenase) denature slowly even at the physiological temperature (37 °C). In the living organism components of the cell, especially the high protein concentration, act as stabilizers, but even there the lifetime of enzymes is limited and they are steadily supplemented by de novo synthesis ( Hinkson and Elias, 2011). To establish the appropriate assay temperature for a distinct enzyme, the temperature dependence of its activity must be analysed.