Enzyme activities detection

The principle of the double mediator mechanism used for intracellular NQO enzyme activity detection is shown in Fig. 11. 

Since endosulfan is a cytochrome P450-dependent monooxygenase inducer, the quantification of specific enzyme activities (e.g., aminopyrine-A -demethylase, aniline hydroxylase) may indicate that exposure to endosulfan has occurred (Agarwal et al. 1978). Because numerous chemicals and drugs found at hazardous waste sites and elsewhere also induce hepatic enzymes, these measurements are nonspecific and are not necessarily an indicator solely of endosulfan exposure. However, these enzyme levels can be useful indicators of exposure, together with the detection of endosulfan isomers or the sulfate metabolite in the tissues or excreta. 

Figure 36-4. Illustration of the tight correlation between the presence of RNA polymerase II and RNA synthesis. A number of genes are activated when Chirono-mus tentans larvae are subjected to heat shock (39 °C for 30 minutes). A Distribution of RNA polymerase II (also called type B) in isolated chromosome IV from the salivary gland (at arrows). The enzyme was detected by immunofluorescence using an antibody directed against the polymerase. The 5C and BR3 are specific bands of chromosome IV, and the arrows indicate puffs. B Autoradiogram of a chromosome IV that was incubated in H-uridine to label the RNA. Note the correspondence of the immunofluorescence and presence of the radioactive RNA (black dots). Bar = 7 pm. (Reproduced, with permission, from Sass H RNA polymerase B in polytene chromosomes. Cell 1982 28 274. Copyright 1982 by the Massachusetts Institute of Technology.)...

PG activity was assayed from cells grown in a medium containing 1% glucose in one-litre self-induced anaerobic fermentation for 5 days by increase of reducing sugars. Enzyme activity increased from pH 3.0 to pH 5.0 (citrate buffer) and decreased drastically above 5.0 (phosphate buffer), but activity was not affected differentially by the two buffers used (data not shown). PG activity increased almost linearly from 20°C to 40°C but above this optimum, activity was lost rapidly and the enzyme was completely inactivated at 60°C and 70°C after 10 and 6 min, respectively (data not shown). No PL, PGL or PME were detected. 

Polygalacturonases were strongly induced in FORL by apple pectin, citric pectin and polygalacturonic acid. When FORL was grown on ucose as carbon source no extracellular PG activity could be detected. Galacturonic acid did not look like a good inducer of PG since the increase of enzyme activity was delayed in comparison with the other carbon sources, after the 5th day of incubation, and also showed lower values (fig.l). 

A large number of amino acid transporters have been detected by isolating mutations which selectively inactivate one permease without altering enzyme activities involving the corresponding amino acid. Competitive inhibition, kinetics and regulatory behaviour have also been used as criteria to distinguish one transport system from another (see section 4.2). 

Monitoring enzyme catalyzed reactions by voltammetry and amperometry is an extremely active area of bioelectrochemical interest. Whereas liquid chromatography provides selectivity, the use of enzymes to generate electroactive products provides specificity to electroanalytical techniques. In essence, enzymes are used as a derivatiz-ing agent to convert a nonelectroactive species into an electroactive species. Alternatively, electrochemistry has been used as a sensitive method to follow enzymatic reactions and to determine enzyme activity. Enzyme-linked immunoassays with electrochemical detection have been reported to provide even greater specificity and sensitivity than other enzyme linked electrochemical techniques. 

The simplest method of coupling enzymatic reactions to electrochemical detection is to monitor an off-line reaction using FIAEC or LCEC. The enzymatic reaction is carried out in a test tube under controlled conditions with aliquots being taken at timed intervals. These aliquots are then analyzed for the electroactive product and the enzyme activity in the sample calculated from the generated kinetic information. 

In addition to enzyme activity, the concentration of an nonelectroactive substrate can be determined electrochemically by this technique. By keeping the substrate (analyte) the limiting reagent, the amount of product produced is directly related to the initial concentration of substrate. Either kinetic or equilibrium measurements can be used. Typically an enzyme which produces NADH is used because NADH is readily detected electrochemically. Lactate has been detected using lactate dehydrogenase, and ethanol and methanol detected using alcohol dehydrogenase... 

Lequea et al. used the activity of tyrosine apodecarboxylase to determine the concentration of the enzyme cofactor pyridoxal 5 -phosphate (vitamin B6). The inactive apoenzyme is converted to the active enzyme by pyridoxal 5 -phosphate. By keeping the cofactor the limiting reagent in the reaction by adding excess apoenzyme and substrate, the enzyme activity is a direct measure of cofactor concentration. The enzymatic reaction was followed by detecting tyramine formation by LCEC. The authors used this method to determine vitamin B6 concentrations in plasma samples. 

Enzyme activity can indicate the exposure of honey to heating and long storage. This criterion is not more accurate than the HMF content value because enzyme activities vary with honey samples. The diastase activity is usually associated with heat treatment. However, its activity gives only an indication about the processing (heat treatment) of the honey but is not suitable for the detection of the origin. 

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