Inactivation rate

M NaCl was 15 hr at 0°C and 45 min at 23°C. Based on this inactivation rate, it is estimated that the purified protein contained only 5-10% of the active form. No method has been found to stabilize the photoprotein. 

Fibrinolytics. Figure 4 Inactivation of plasmin by a2-plasmin inhibitor Effect of fibrin. The inactivation rate of free plasmin is very rapid (the second order rate constant k 430 104M-1s-1), while of fibrin bound plasmin is slow (the second order rate constant k 1 104M"1s"1). Inactivation of plasmin in the figure is shown in arbitrary units. Abbreviations plasmin (P), fibrin (F). 

Fig. 27. The effects of shear on the inactivation rate of lactate dehydrogenase (LDH) at different conditions. LDH are subjected to shear at a mean velocity gradient of 6490 s at 30 °C [107]...

The inactivation is normally a first-order process, provided that the inhibitor is in large excess over the enzyme and is not depleted by spontaneous or enzyme-catalyzed side-reactions. The observed rate-constant for loss of activity in the presence of inhibitor at concentration [I] follows Michaelis-Menten kinetics and is given by kj(obs) = ki(max) [I]/(Ki + [1]), where Kj is the dissociation constant of an initially formed, non-covalent, enzyme-inhibitor complex which is converted into the covalent reaction product with the rate constant kj(max). For rapidly reacting inhibitors, it may not be possible to work at inhibitor concentrations near Kj. In this case, only the second-order rate-constant kj(max)/Kj can be obtained from the experiment. Evidence for a reaction of the inhibitor at the active site can be obtained from protection experiments with substrate [S] or a reversible, competitive inhibitor [I(rev)]. In the presence of these compounds, the inactivation rate Kj(obs) should be diminished by an increase of Kj by the factor (1 + [S]/K, ) or (1 + [I(rev)]/I (rev)). From the dependence of kj(obs) on the inhibitor concentration [I] in the presence of a protecting agent, it may sometimes be possible to determine Kj for inhibitors that react too rapidly in the accessible range of concentration. ... 

Despite a higher intrinsic reactivity, epoxides of type 35 and 36 show a lower inactivation rate kj(max), as seen in Table XI, than the conduritol epoxides. This is probably caused by the greater flexibility of the epoxyalkyl chain in the active-site cleft, and by non-productive binding in positions where the oxirane is not within reach of the catalytic groups of the active site. For epoxypropyl oligosaccharides, this would hold even when the inhibitor occupies the correct subsites. 

The pH-dependence of the inactivation rate indicated the participation of both a basic and an acidic group in the reaction with 40. The latter could be explained by the formation at the active site of the highly reactive epoxide 1,2-anhydroconduritol F (42) which is subsequently activated by the acidic... 

The third is the effect of temperature as typically the inactivation rate increases/decreases with temperature for gram-positive/gram-negative bacteria. The exception to this rule are coliforms. All microorganisms display, in any case, very narrow temperature ranges where photocatalytic disinfection activity reaches maximum values. ... 

Dekker et al. [170] studied the extraction process of a-amylase in a TOMAC/isooctane reverse micellar system in terms of the distribution coefficients, mass transfer coefficient, inactivation rate constants, phase ratio, and residence time during the forward and backward extractions. They derived different equations for the concentration of active enzyme in all phases as a function of time. It was also shown that the inactivation took place predominantly in the first aqueous phase due to complex formation between enzyme and surfactant. In order to minimize the extent of enzyme inactivation, the steady state enzyme concentration should be kept as low as possible in the first aqueous phase. This can be achieved by a high mass transfer rate and a high distribution coefficient of the enzyme between reverse micellar and aqueous phases. The effect of mass transfer coefficient during forward extraction on the recovery of a-amylase was simulated for two values of the distribution coefficient. These model predictions were verified experimentally by changing the distribution coefficient (by adding... 

Dekker et al. [170] have also shown that the steady state experimental data of the extraction and the observed dynamic behavior of the extraction are in good agreement with the model predictions. This model offers the opportunity to predict the effect of changes, both in the process conditions (effect of residence time and mass transfer coefficient) and in the composition of the aqueous and reverse micellar phase (effect of inactivation rate constant and distribution coefficient) on the extraction efficiency. A shorter residence time in the extractors, in combination with an increase in mass transfer rate, will give improvement in the yield of active enzyme in the second aqueous phase and will further reduce the surfactant loss. They have suggested that the use of centrifugal separators or extractors might be valuable in this respect. 

Disraption of side chain interactions results in denaturation of the protein, and the rate of inactivation follows first order kinetics in the simplest cases. The plot of the logarithm of the remaining activity (In a ) versus time gives a straight line, the slope of which is the negative value of the inactivation rate constant. 

At low temperature (T and T2 in Figure 8.3) the rate of heat inactivation is slow as compared to the rate of the catalysed reaction. At elevated temperature (T3, T ) the increased heat-inactivation rate results in a faster decrease in the number of the active catalyst molecules. As a consequence of this the rate of the enzymic reaction becomes... 

By comparing interfacial inactivation rates in a stirred-cell (low and controlled area of exchange) and an emulsion system (high interfacial area), these authors have shown that the use of an emulsion system can be exploited to obtain high solute interphase mass-transfer rates since the rate of specific interfacial inactivation remains low. However, in this system, the presence of an epoxide substrate at high concentration in the organic phase increases the rate of interfacial inactivation. Addition of a sacrificial protein to the system, which can prevent adsorption of the catalytic enzyme at the interface, could provide a method to reduce the rate of interfacial inactivation. 

An enzyme is irreversibly heat inactivated with an inactivation rate of k = 0.001 s at 80 °C. Estimate the half-life t of this enzyme at 80 °C. 

S.I. Hong et al. [42] confirmed that the inactivation rate increased with pressure, exposuretime and with decreasing pH of media. They stated that microbial inactivation was governed essentially by penetration of CO2 into cells, and its effectiveness could be improved by enhancing the transfer rate. Microbial reduction of more than six powers of ten occurred within 30 min, under a CO2 pressure of 2000 psi at 30°C. The authors hypothesized that cell death resulted from the lowered intracellular pH and damage to the cell membrane owing to penetration of CO2. 

Solutions of acid phosphatase are particularly sensitive to surface inactivation. Figure 3 (88) shows the inactivation rate of the enzyme in the presence and absence of surface-active detergents. The inactivation process is temperature sensitive and the protection by detergent is total. Most of the enzyme inactivation proceeds with first-order kinetics. A variety of agents—gelatin, bovine serum albumin, egg albumin, and Tween-80—protect the enzyme against inactivation. 

Fig. 3. Surface inactivation rate of prostatic acid phosphatase by shaking and protection by added surface-active agent. Shaking mixtures (20 ml) contained purified enzyme (056 /ug of protein/ml) in 0.05 M acetate buffer at pH 5.5. The solutions were shaken in 50 ml volumetric flasks using a mechanical shaker (Burrell, model CC). Temperatures were maintained by immersion of the flasks in an appropriately set water bath. After specified intervals of shaking, duplicate 0.1 ml ahquots were removed into tubes containing Triton X-100. All tubes were assayed simultaneously, following the shaking procedure, with 0.05 M phenyl phosphate as substrate. Curve 1 Enzyme + Triton X-100 at 0°C and 29°C. Curve 2 Enzyme alone at 0°C. Curve 3 Enzyme alone at 29°C. From Tsuboi and Hudson (88).

The rate of inactivation by chelators is strongly dependent on temperature, pH, and protein concentration (13). Between 16° and 30° the activation energy for the chelator-dependent loss of activity is 41 kcal at pH 8.2. At 30° the rate of inactivation is over 200-fold faster at pH 8.7 than at pH 7.2. The inactivation is much faster in dilute than in concentrated enzyme solutions as the protein concentration is increased, correspondingly more rigorous conditions must be employed to observe inactivation. The rate of inactivation appears to exhibit saturation kinetics with respect to chelator concentration. At high EDTA levels the inactivation rate approaches a maximum which is independent of chelator concentration (13). 

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