However, in many cases irreversible inactivation processes (which may involve a reversibly unfolded form as an intermediate) occur on a timescale comparable with that of the assay. Under these circumstances the reaction progress at higher temperatures is strongly curved, as enzyme is inactivated. Then it is difficult to estimate a meaningful

initial rate. Some studies will define activity based on a single time point measurement of product formed (or substrate consumed). In studies of temperature effects this is a particularly dangerous design. With progress Talazoparib curve in reality strongly curved, the estimate of “activity” (based on an assumption of linear progress) will be higher the shorter the choice of reaction time. As temperature increases, the rate at the shortest times may continue to increase due to normal thermal effects, but faster inactivation will increase curvature of progress. Hence the apparent “optimum temperature” will depend on the arbitrary choice of assay duration, being highest for the shortest assays. • It is necessary that the buffer in which the thermal exposure is carried out is described completely. Ionic strength may play a role (see also Bisswanger,

2014). Presence of additives can significantly affect the temperature optimum. This includes presence of simple ions. Calcium ion, for example, affects both the activity selleck kinase inhibitor and/or stability of several enzymes. Thermal stability is the most frequently studied parameter in order to assess the stability of the enzyme in general terms. It is not an incorrect trend in as much as a more thermostable enzyme is more likely to be stable under other harsh conditions as well, for example, when exposed to organic solvents. The inactivation mechanisms of an enzyme under all

conditions involve presumably unfolding of the protein chain as the first common step (Gupta, 1993). However, in recent years, “native-like structures” are known to aggregate (Bemporad et al., 2012). At the same time, aggregation need not result in inactivation. As already mentioned, we have recently reported an aggregated form of α-chymotrypsin which shows higher activity in both aqueous buffers and non-aqueous media (Rather et al., 2012). Stabilization under extreme pH conditions is also a desirable goal in several cases. Stability of proteases Oxymatrine under alkaline conditions, for example, is useful for incorporating these enzymes in detergents. Often, such stability or stabilization is reported when the biocatalyst prepared is dissolved or suspended in aqueous buffers. In terms of validity of the data, that is not a problem provided all conditions are properly defined. This is necessary since for a protein solution, stability strongly depends upon the concentration, the nature of the buffer and the presence of any other additive. From practical point of view, such data merely provides a rough guideline.