Recovery of activity

The bacterial culture converts a portion of the supplied nutrient into vegetative cells, spores, crystalline protein toxin, soluble toxins, exoenzymes, and metabolic excretion products by the time of complete sporulation of the population. Although synchronous growth is not necessary, nearly simultaneous sporulation of the entire population is desired in order to obtain a uniform product. Depending on the manner of recovery of active material for the product, it will contain the insolubles including bacterial spores, crystals, cellular debris, and residual medium ingredients plus any soluble materials which may be carried with the fluid constituents. Diluents, vehicles, stickers, and chemical protectants, as the individual formulation procedure may dictate, are then added to the harvested fermentation products. The materials are used experimentally and commercially as dusts, wettable powders, and sprayable liquid formulations. Thus, a... 

If the inhibitor is found to bind rapidly (linear progress curves) and dissociate rapidly (rapid recovery of activity upon dilution) from its target enzyme, then one can proceed to analyze its inhibition modality and affinity by classical methods. The modes of reversible inhibition of enzymes were described in Chapter 3. In the next section of this chapter we will describe convenient methods for determining reversible inhibition modality of lead compounds and lead analogues during compound optimization (i.e., SAR) studies. 

Simple removal of the bead(s) from the reaction is enough to eliminate the iodination process. The mild nature of the Iodobeads iodination reaction can result in better recovery of active protein than using soluble oxidants (Lee and Griffiths, 1984). 

Figure 2.12. Recovery of Active Rhodium from a Spent Catalyst Solution...

Initial stages in the production of murine monoclonals entail administration of the antigen of interest to a mouse. This is followed by sacrifice and recovery of activated B-lymphocytes from the spleen. A similar approach to the production of human monoclonals would be unethical. Administration of some antigens to humans could endanger their health. Although B-lymphocytes could be obtained from the peripheral circulation, the majority of these are unstimulated, and recovery of (stimulated) B-lymphocytes from the spleen is impractical. 

Figure 8.2 The effect of pH on the enzyme lactate dehydrogenase (EC 1.1.1.27). The enzyme shows maximum activity at pH 7.4 (A). When stored in buffer solutions with differing pH values for 1 h before re-assaying at pH 7.4, it shows complete recovery of activity from pH values between 5 and 9 but permanent inactivation outside these limits (B).

Because cholinesterase inhibition is a very sensitive biomarker for other chemicals, it is not always conclusive evidence of disulfoton exposure. However, depression of cholinesterase activity can alert a physician to the possibility of more serious neurological effects. Erythrocyte acetylcholinesterase activity more accurately reflects the degree of synaptic cholinesterase inhibition in nervous tissue, while serum cholinesterase activity may be associated with other sites (Goldfrank et al. 1990). In addition, a recent study showed that after rats received oral doses of disulfoton for 14 days, acetylcholinesterase levels in circulating lymphocytes correlated better with brain acetylcholinesterase activity than did erythrocyte cell cholinesterase activities during exposure (Fitzgerald and Costa 1993). However, recovery of the activity in lymphocytes was faster than the recovery of activity in the brain, which correlated better with the activity in erythrocytes. Animal studies have also demonstrated that brain acetylcholinesterase depression is a sensitive indicator of neurological effects (Carpy et al. 1975 Costa et al. 1984 Schwab and Murphy 1981 Schwab et al. 1981, 1983) however, the measurement of brain acetylcholinesterase in humans is too invasive to be practical. 

The schematic below shows that in this dynamic system PAN could affect the synthetic process (site 1), the enzyme itself (site 2), or the degradation process (site 3). If the site of attack were site 2, the synthetic process might compensate for degradation of the enzyme by producing more. If the enzyme activity were measured as a function of time after exposure, there would be first a decrease and then recovery of activity. (Such a response has been observed for the effect of ozone on respiration.) Effects at site 3 would show first an increase in activity and en, if the system were regulated, a decline to normal, as the synthetic process slowed down. Effects at site 1 would cause a decrease in activity commensurate with the rate of enzyme degradation. 

Fm-thermore, there is a good correlation between recovery of activity and ability to crystallize. Thus, particles lacking protein BL12, which are inactive biologically, could not be crystallized, whereas the reconstituted particles produce crystals isomorphous with the native form. 

Zinc not only provides protection, but can gradually reverse spontaneous inactivation, as illustrated in Fig. 5. The enzyme was incubated at 37°, and zinc sulfate was added after 3 hours to cause a progressive recovery of activity. The slow reactivation is in contrast to the instantaneous effect of Zn2+ on an EDTA-treated, enzyme preparation. In a preparation that had lost activity during storage without the addition of Zn2+, the gradual increase in activity on incubation with Zn2+ frequently led to a value greater than that of the unincubated control. 

No. Name of Active Material Concentration of Standard Solution Method Used for Testing % Recovery of Active Ingredient % Recovery per Limit (95-105%)... 

The enzyme, monoamine oxidase, exists in two forms MAO-A (intestinal mucosa and intraneuronally in the brain) and MAO-B (platelets and mainly extraneuronally in the brain). Serotonin is preferentially metabolised by MAO-A and noradrenaline (NA norepinephrine), and dopamine and lyramine by both forms. The first generation MAOI antidepressants (phenelzine, tranylcypromine, and isocarboxazid) inhibit both MAO-A and MAO-B and are thought to work by increasing the availability of 5-HT and NA in the synapse—with longer-term adaptive effects occurring as for the TCAs. These MAOls are irreversible, i.e. they permanently inactivate MAO. Thus, recovery of activity occurs slowly, over days, as new MAO molecules are synthesised. 

The Sheldon group [87] prepared aquagels of different HNLs and compared them in the synthesis reaction of different cyanohydrins with the CLEAs and the free enzymes. The activity recovery for the aquagels and CLEAs measured by a photometric assay were quite low. Using the same loadings, the aquagels turned out to be much faster than the free enzyme. This confirms the underestimation of the recovery of activity by fast assays due to diffusion problems, as reported earlier [74, 75]. The stability and the catalytic performance of the immobilized HNLs are strongly influenced by the solvent, the immobilization method, and the enzyme source. 

Nine determinations total, three concentrations, three separately prepared replicates of each. Range of 80 to 120% of the nominal concentration determine the recovery of active sample from the inert matrix. 

Cloetens (98) dialyzed pig kidney phosphatase against 0.01 M KCN for 6 days and found a considerable loss in activity. However, several minutes preincubation with Mg2+ before assay gave up to 40% recovery of activity. Of a series of metal iqns tested, Zn2+ was the most effective giving 70% recovery. Hofstee investigated the effects of glycine, EDTA, and metal ions on calf intestinal phosphatase (99) and concluded that dialysis against EDTA produced an inactive enzyme. Addition of Zn2+... 

As shown in the preceding section, in the presence of 5 M guanidine hydrochloride the six subunits of the enzyme were separated from one another and opened up to form randomly coiled, single polypeptide chains. These were catalytically inactive (13). When the guanidine hydrochloride was removed by dialysis, enzymic activity was restored, provided the protein was in a reducing environment during the dialysis (Table II). Under optimal conditions (No. 5 of Table II) there was 80-90% recovery of activity. If there was opportunity for persistent disulfide... 

No. Denaturation treatment1 Renaturation treatment1 Specific activity (units/mg protein) Recovery of activity (%)... 

Reports on the action of reduced glutathione give results quite different from the above. The bulky mercaptan does not reduce RNase at all at room temperature. As the thermal transition temperature is approached the reduction appears to be all or none with no evidence of partially reduced intermediates 186). In a study of the recovery of activity after denaturation in 8 M urea, Kim and Paik 187) reported... 

IODO-BEADS reaction from the manufacturer s recommended pH 7.0 to 8.2 (Tsomides et al., 1991). No reducing agent is required to stop the iodination reaction, as is the case with chloramine-T and other methods. Simple removal of the bead(s) from the reaction is enough to eliminate the iodination process. The mild nature of the IODO-BEADS iodination reaction can result in better recovery of active protein than using soluble oxidants (Lee and Griffiths, 1984). 

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