Recovery of Enzyme Activity

In order to compare the effectiveness of different methods of immobilization it is useful to determine the portion of enzyme remaining 

The remanent activity of glucose oxidase (GOD) membranes has been determined by measuring the initial rate of H2O2 formation with a Pt electrode polarized to +600 mV, using a known amount of resolubilized membrane in 5 mmol/1 glucose solution at 37°C (Scheller et al., 1988). Thus, 70-90% of the initial activity has been found with gelatin-entrapped enzyme. The GOD membrane was solubilized in 0.05 mol/1 phosphate buffer, pH 5.5, at 40°C. 

The determination of the initial rate of product formation or substrate consumption in the measuring cell using the intact membrane or the complete sensor gives a measure of the enzyme activity acting in the measuring process. By comparison with the residual activity the excess of enzyme may be estimated. 

With a gelatin membrane entrapped between two dialysis membranes and containing 46 U/cm2 of enzyme, the H2O2 formation corresponds to only 110 mU/cm2, i.e., less than 1% of the initial enzyme activity (Fig. 28). This 

In Table 5 the apparent activities of enzymes entrapped in or covalently fixed to membranes are compared to those of enzymes directly adsorbed or fixed to electrode surfaces. It may be concluded that different immobilization procedures lead to approximately identical apparent activities. The advantage of direct attachment to the transducer surface is the low diffusion resistance of the monomolecular enzyme layer. On the other hand, enzyme membranes are more stable because of their inherent enzyme excess. 

---

Figure 5.9 Recovery of enzyme activity after rapid dilution as described in Figure 5.8. Curve a represents the expected behavior for a control sample that was pre-incubated and diluted in the absence of inhibitor. Curve b represents the expected behavior for a rapidly reversible inhibitor. Curve c represents the expected behavior for a slowly reversible inhibitor, and curve d represents the expected behavior for an irreversible or very slowly reversible inhibitor. See color insert.

The initial hydroxylation of tryptophan, rather than the decarboxylation of 5-HTP, appears to be the rate-limiting step in serotonin synthesis. Therefore, the inhibition of this reaction results in a marked depletion of the content of 5-HT in brain. The enzyme inhibitor most widely used in experiments is parachlorophenylalanine (PCPA). In vivo, PCPA irreversibly inhibits tryptophan hydroxylase, presumably by incorporating itself into the enzyme to produce an inactive protein. This results in a long-lasting reduction of 5-HT levels. Recovery of enzyme activity, and 5-HT biosynthesis, requires the synthesis of new enzyme. Marked increases in mRNA for tryptophan hydroxylase are found in the raphe nuclei 1-3 days after administration of PCPA [6]. 

A (rapidly) reversible inhibitor will permit rapid and complete recovery of enzyme activity by dialysis. However, irreversible inhibitors are not removed by this procedure. Recovery from tight-binding inhibition is usually slow it is not uncommon for several dialysis bags containing enzyme to be prepared and for activity in each to be determined at various time points following the commencement of dialysis. The off-rate of these inhibitors is generally more rapid at higher temperatures. 

Any substance that can diminish the velocity of an enzyme-catalyzed reaction is called an inhibitor. Reversible inhibitors bind to enzymes through noncovalent bonds. Dilution of the enzyme-inhibitor complex results in dissociation of the reversibly bound inhibitor, and recovery of enzyme activity. Irreversible inhibition occurs when an inhibited enzyme does not regain activity on dilution of the enzyme-inhibitor complex. The two most commonly encountered types of inhibition are competitive and noncompetitive. 

EN129 Saini, M., Brooks, E., Cassano, T., Daggett, S., Fishers, D., Greenberg, N., Mowers, P. and Van Brunt, N. (1992). Stability and recovery of enzyme activity in calibrators for Kodak Ektachem analyzers. Clin. Chem. 38,981, Abstr. 196. 

Route of exposure may have much to do with the recovery from OPs. When pigeons were treated orally with an OP, inhibition of blood ChE was rapid, and recovery of activity occurred within a few days. However, when the treatment was conducted der-mally, putting the OP on the feet, recovery of enzyme activity took several weeks, implying the presence of a depot for OPs and the possibility that birds can accumulate OPs by flying from site to site. The possibility of bioaccumulation of OPs in a food chain (usually considered to be a characteristic of chlorinated hydrocarbons) was demonstrated by the report of an eagle poisoned by an OP (Warbex) in magpies that, in turn, had obtained the OP by ingesting hair from a steer that had been treated with it for parasites. 

Deoxy-2-fluoro-p-glycosyl fluorides identified the nucleophiles in several enzymes, such as sweet almond p-glucosidase (GH 1) " and human p-glucuronidase (GH 2) and in GH 30. " When they were tested in vivo, the activity recovered much faster than when the same enzyme was inactivated by the appropriate triazene. Recovery of enzyme activity in the triazene case was associated with new protein synthesis, but 2-fluoro-2-deoxyglycosyl enzymes slowly reverted to the original active enzyme. ... 

Characterization of Immobilized Enzymes in Biosensors 14.5.2.1 Recovery of Enzyme Activity... 

The enzyme purification protocol is described in Table 1. The overall recovery of enzyme activity was 15% with an 85 fold purification. There was an increase in... 

Carbamate poisoning produces reversible acetylcholinesterase inhibition, and spontaneous recovery of enzyme activity may occur within several hours, making these tests less usehl. 

Recovery of Enzyme Activity following High-Perfonnaoce HydropboUc Interaction Chromatography ... 

In the first instance, the inhibitor reacts with the serine hydroxyl moiety of the enzyme to produce the alkylphosphate ester [14]. The formation of this ester is reversible and the enzyme activity may be recovered by nucleophilic attack with compounds such as hydroxylamine [15] and oximes [16]. Alternatively, dealkylation of the enzyme-phosphate may then occur [17], a process known as ageing [18], and the covalent link between the enzyme and the phosphate is stabilised, so preventing any recovery of enzyme activity by oximes [19]. 

Nitrite reductase has been extensively purified and characterized from a diverse group of photosynthetic organisms (Hattori and Uesugi, 1%8 Cardenas et al., 1972 Zumft, 1972 Ho and Tamura, 1973 Hucklesby et al., 1976 Vega and Kamin, 1977). The most extensive purification with a relatively high recovery of enzyme activity utilized affinity chromatography on ferredoxin-Sepharose (Ida, 1977). 

Zieve, L., Doizaki, W. M., and Stenroos, L. E., 1968a, Effect of magnesium deficiency on blood and liver transketolase activity and on the recovery of enzyme activity in thiamine-deficient rats receiving thiamine, J. Lab. Clin. Med. 72 268. 

The off rate, kos, of the inhibition can be determined by calculation using Eq. (13.6) or by direct measurement. Enzyme-inhibitor complex can be isolated from excess inhibitor by size exclusion chromatography, preferably with a shift in pH to a range where the enzyme is stable but inactive, to stabilize the complex (Copp et al., 1987). It can then be added back to an activity assay, to measure the return of enzyme activity over time. The recovery of enzyme activity, koff, should be a first-order process, independent of inhibitor, enzyme, or E-I concentrations. The final rate, C, will depend on [E-I] (and any free E that might have been carried through the chromatography). 

Enzyme activities were determined using established methods from the literature and were optimized to give the maximum rate. The recovery of enzyme activity in the washed plastids was determined as a percentage of that in the initial homogenate. The latency of enzyme activity was determined by assaying the enzyme activities using intact plastids under iso-osmotic conditions, and using plastids that had been osmotically ruptured. 

---