Inhibitors, enzyme sensors

Enzyme Sensors for Inhibitors - Toxic Effect Sensors... 

Enzyme/indicator optrodes for registration of enzyme reactions and their inhibitors, such as heavy metal ions and pesticides, can be produced by PESA technique. Composite films containing the enzyme urease and cyclo-tetra-chromotropylene as indicator molecules show some characteristic spectral transformations caused by urea decomposition. The reaction of inhibition of urease by heavy metal ions can be also registered with this method. Further development of the enzyme sensors and sensor arrays lies in finding suitable pairs of enzyme/indicator, their deposition by PESA method and studying the enzyme reactions (including inhibition) with UV-vis spectroscopy. 

The response of an enzyme sensor in the steady state depends largely on the ratio of the substrate concentration [5] to the enzyme Michaelis constant K. When [S K is large, the reaction rate reaches a maximal value V,, which is proportional to the number of active sites of the immobilized enzyme. The reaction rate is independent of the substrate concentration, and the product concentration at the contact with the electrode is the same for all high substrate concentration. The quantify of enzyme in the layer determines the linear zone in the response to the substrate concentration. This zone corresponds to first-order kinetics with respect to substrate concentration, whereas the region with a plateau has zeroth-order kinetic. When the substrate concentration is very high([5] K ), the biosensor is no longer capable of determining the substrate but may determine inhibitors which affect the minimal rate of the enzymatic reaction... 

When an enzyme sensor is intended to determine a species that inhibits an enzymatic reaction, the substrate concentration is fixed at a given value, and the inhibitor concentration is varied. In this case, it is possible to represent both the variation in potential and the variation corresponding to the percentage inhibition (/%) ... 

Evtugyn GA, Budnikov HC, Nikolskaya EB (1998) Sraisitivity and selectivity of electrochemical enzyme sensors for inhibitor determinatirai. Talanta 46(4) 465-484. doi 10.1016/ S0039-9140(97)00313-5... 

An enzyme sensor can be considered as the combination of a transducer and a thin enzymatic layer, which normally measures the concentration of a substrate. The enzymatic reaction transforms the substrate into a reaction product that is detectable by the transducer. An extension of this definition is that the concentration of any substance can be measured provided that its presence affects the rate of an enzymatic reaction this is especially true for enzyme inhibitors. We will first examine the principle of enzyme sensors and the theoretical and practical aspects of their operation. We will then describe the various sensors, classified according to their detection mode. 

Inside living cells, enzymes exist in an extremely structured environment, which may have the form of a gel, a membrane or other solid state assemblies. Under these conditions, the behavior of immobilized enzymes towards their inhibitors is very different from that in the homogeneous phase. The mode of enzymatic inhibition is variable from one inhibitor to another, and may also be reversible or irreversible. Inhibition may be observed with enzyme sensors without having to resort to techniques that require long extractions, such as dialysis or gel filtration, which are normally used to separate an inhibitor from a soluble enzyme. The response of the enzyme sensor provides a continuous measure of the activity of the immobilized enzyme, whether or not its inhibitor is present. Enzyme sensors thus allow convenient and rapid study of the inhibition of enzymes and their subsequent reactivation. 

When the enzyme sensor is in contact with an inhibitor, the reaction rate diminishes according to the inhibition mode involved. The substrate and product concentrations in the steady state can be obtained by solving nations (3) and (4), provided that V , and are replaced by Vp and Kp. Figure 4.5 shows the concentration profiles of the substrate (broken line) and the product (solid line) in terms of K ,. 

The digital simulation above gives the concentration of the substrate [S]g and, more importantly, that of the product [P]e at the [Pig-sensitive base transducer. Using this information, the response of the enzyme sensor in the presence of different inhibitor concentradons expressed in... 

Once the enzyme sensor has been in contact with the inhibitor, it is rinsed with a solution containing a reactivating agent. In principle, the injection of substrate should give the reference peak again. 

Campanella L, Achili M, Sammartino MP, Tomasetti M (1991) Butyrylcholine enzyme sensor for determining organophosphoms inhibitors. Bioelectrochem Bioenerg 26 237-249... 

Trettnak W., Reininger F., Zinterl E., Wolfbeis O.S., Fiber Optic Remote Detection of Pesticides and Related Inhibitors of the Enzyme Acetylcholine Esterase, Sensor Actuat B-Chem 1993 11 87. 

Enzymes can be used not only for the determination of substrates but also for the analysis of enzyme inhibitors. In this type of sensors the response of the detectable species will decrease in the presence of the analyte. The inhibitor may affect the vmax or KM values. Competitive inhibitors, which bind to the same active site than the substrate, will increase the KM value, reflected by a change on the slope of the Lineweaver-Burke plot but will not change vmax. Non-competitive inhibitors, i.e. those that bind to another site of the protein, do not affect KM but produce a decrease in vmax. For instance, the acetylcholinesterase enzyme is inhibited by carbamate and organophosphate pesticides and has been widely used for the development of optical fiber sensors for these compounds based on different chemical transduction schemes (hydrolysis of a colored substrate, pH changes). 

In AChE-based biosensors acetylthiocholine is commonly used as a substrate. The thiocholine produced during the catalytic reaction can be monitored using spectromet-ric, amperometric [44] (Fig. 2.2) or potentiometric methods. The enzyme activity is indirectly proportional to the pesticide concentration. La Rosa et al. [45] used 4-ami-nophenyl acetate as the enzyme substrate for a cholinesterase sensor for pesticide determination. This system allowed the determination of esterase activities via oxidation of the enzymatic product 4-aminophenol rather than the typical thiocholine. Sulfonylureas are reversible inhibitors of acetolactate synthase (ALS). By taking advantage of this inhibition mechanism ALS has been entrapped in photo cured polymer of polyvinyl alcohol bearing styrylpyridinium groups (PVA-SbQ) to prepare an amperometric biosensor for... 

In addition to hydrogen ions, other species can also affect the enzymatic catalytic activity. This phenomenon is called inhibition it may be specific, nonspecific, reversible, or irreversible. The inhibition reactions can also be used for the sensing of inhibitors. The best-known example is the sensor for detection of nerve gases. These compounds inhibit the hydrolysis of the acetylcholine ester which is catalyzed by the enzyme acetylcholine esterase. Acetylcholine ester is a key component in the neurotransmission mechanism. 

With regard to assaying the inhibitory activity of extracts electro-chemically, one of the problems of assays using sensors based on cholinesterase was that considerable time, e.g. 30-45 min [46,47], could be needed for the activity of the enzyme electrode to fall below control levels. The time increased as the level of inhibition decreased. Such lengthy assays make any number of serial assays impractical. In previous work [48,49], it had been noted that if sensors were exposed to solution containing inhibitors and then allowed to dry, they could be... 

Photoswitchable enzymes could have an important role in controlling biochemical transformations in bioreactors. Various biotechnological processes generate an inhibitor, or alter the environmental conditions (pH, for example) of the reaction medium. Photochemical activation of enzymes that adjust environmental conditions or deplete the inhibitor to a low concentration may maintain the bioreactor at optimal performance. More specifically, integration of the photoswitchable biocataly-tic matrix with a sensory electrode might yield a feedback mechanism in which the sensor element triggers the light-induced activation/deactivation of the photosensitive biocatalyst. 

An alternative approach, adopted by Albery et al. [59-61], is to determine the mechanism giving rise to the sensor response and to use this information together with the measured data at short times to calculate the final response. This was used for an electrochemical sensor system incorporating cytochrome oxidase where the steady-state responses of the measurement system were insufficiently fast for useful measurement of respiratory inhibitors such as cyanide, hydrogen sulphide, etc. By using mechanistic information, it was possible to successfully calculate the concentration in a test sample by real-time analysis of the sensor signals at short times after exposure to the test sample. The analysis could cope with the gradual loss of enzyme activity commonly found in these biosensor devices. 

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