Enzyme mediator-modified

Fig. 8. Entrapment of mediator-modified enzymes within a conductive polymer film where ( ) represents the mediator ferrocene and (B) the active site...

The pioneering works of Hill and Eddows have opened the way to realize fast and efficient electron transfer of enzymes at the electrode surface. They modified a gold electrode with 4,4 -bipyrydyl, an electron promoter, not a mediator since it does not take part in electron transfer in the potential region of interest, to accomplish rapid electron transfer of cytochrome [1], Their work has triggered intensive investigation of electron transfer of enzymes using modified electrodes [2]. 

Electron mediator Either electrode of enzyme is modified by an electron mediator in various manners. 

Self-Assembly of Mediator-Modified Redox Enzymes on the Porous Gold Electrode Surface... 

In contrast to the molecular wire of molecular interface, electron mediators are covalently bound to a redox enzyme in such a manner as an electron tunneling pathway is formed within the enzyme molecule. Therefore, enzyme-bound mediators work as molecular interface between an enzyme and an electrode. Degani et al. proposed the intramolecular electron pathway of ferrocene molecules which were covalently bound to glucose oxidase [ 4 ]. However, few fabrication methods have been developed to form a monolayer of mediator-modified enzymes on the electrode surface. We have succeeded in development of a novel preparation of the electron transfer system of mediator-modified enzyme by self-assembly in a porous gold-black electrode as schematically shown in Fig.12 [14]. 

Fig.12 Scheme of self-assembly of mediator-modified enzyme on the gold electrode surface... 

To overcome the poor stability of ferrocene-mediated enzyme sensors, mediator-modified electrodes have been used. In the case of glucose oxidase, the cofactor FAD is deeply buried within the protein matrix. The depth of the active center is estimated to be 0.87 nm. Therefore, one cannot expect that the mediator covalently attached to the electrode surface via a short spacer retain the possibility of closely approaching the cofactor of the enzyme. 

In contrast to the mediator-modified electrodes, Degani et al. modified glucose oxidase itself by means of covalently bound ferrocene [4]. After modifying enzymes with ferrocene carboxylic acid, they observed direct electron transfer from the active site of the enzyme to a gold or platinum... 

Classical bacterial exotoxins, such as diphtheria toxin, cholera toxin, clostridial neurotoxins, and the anthrax toxins are enzymes that modify their substrates within the cytosol of mammalian cells. To reach the cytosol, these toxins must first bind to different cell-surface receptors and become subsequently internalized by the cells. To this end, many bacterial exotoxins contain two functionally different domains. The binding (B-) domain binds to a cellular receptor and mediates uptake of the enzymatically active (A-) domain into the cytosol, where the A-domain modifies its specific substrate (see Figure 1). Thus, three important properties characterize the mode of action for any AB-type toxin selectivity, specificity, and potency. Because of their selectivity toward certain cell types and their specificity for cellular substrate molecules, most of the individual exotoxins are associated with a distinct disease. Because of their enzymatic nature, placement of very few A-domain molecules in the cytosol will normally cause a cytopathic effect. Therefore, bacterial AB-type exotoxins which include the potent neurotoxins from Clostridium tetani and C. botulinum are the most toxic substances known today. However, the individual AB-type toxins can greatly vary in terms of subunit composition and enzyme activity (see Table 2). 

Figure 1 The mode of action for bacterial AB-type exotoxins. AB-toxins are enzymes that modify specific substrate molecules in the cytosol of eukaryotic cells. Besides the enzyme domain (A-domain), AB-toxins have a binding/translocation domain (B-domain) that specifically interacts with a cell-surface receptor and facilitates internalization of the toxin into cellular transport vesicles, such as endosomes. In many cases, the B-domain mediates translocation of the A-domain into the cytosol by pore formation in cellular membranes. By following receptor-mediated endocytosis, AB-type toxins exploit normal vesicle traffic pathways into cells. One type of toxin escapes from early acidified endosomes (EE) into the cytosol, thus they are referred to as short-trip-toxins . In contrast, the long-trip-toxins take a retrograde route from early endosomes (EE) through late endosomes (LE), trans-Golgi network (TGN), and Golgi apparatus into the endoplasmic reticulum (ER) from where the A-domains translocate into the cytosol to modify specific substrates.

The eells were more resistant to the proteolytic enzyme-mediated as well as spontaneous detachment. They were able to persist on the modified polymer surfaees in confluent, viable and metabolically active state for several months [7-11,18,19,40,132-134]. 

Both the determinations reported here rely on the inhibition activity of OPs pesticides toward AChE combined with the detection of the AChE enzyme product choline at the surface of a mediator-modified screen-printed choline oxidase electrochemical biosensor. 

Imamura M, Haruyama T, Kobatake E, Ikariyama Y, Aizawa M. Self-assembly of mediator-modified enzyme in porous gold-black electrode for biosensing. Sensors and Actuators B 1995, 24, 113-116. 

Mediators. Since direct electrode transfer between enzyme and electrode can rarely be performed with repeated efficiency, it was obvious that a synthetic replacement must be found for oxygen, and there thus began the hunt for modified electrodes and enzyme mediators. Many electron acceptor molecules and complexes... 

The lincosamides, lincomycin and clindamycin are active against Grampositive bacteria. Plasmid-mediated inactivation from enzymatic nucleo-tidylation occurs in some staphylococci. Plasmid-encoded enzymes can modify streptogramin A (O-acetyltransferase enzyme) and streptogramin B (hydrolase enzyme involved) in S. aureus [198, 199], There is no evidence that bacteria can circumvent the action of other antibiotics for example, mupirocin is not degraded [200]. 

Ferrocene modified flexible polymeric electron transfer systems Ferrocene and its derivatives are readily available and commonly used organometalUc redox mediators, so it is quite natural that they were selected first to synthesize mediator modified polymeric electron transfer systems. Siloxane pol5uners are flexible but aqueous insoluble pol3nmers. As previously indicated, a flexible polymer backbone allows close contact between the redox center(s) of the enzyme and the mediator, and the water insoluble property of the polymer prevents not only redox polymer from leaching into bulk media but also prevents enzyme diffusion away fi-om the electrode surface by entrapping it in the polymer/carbon paste matrix. Therefore, ferrocene and... 

Kashiwagi, Y., Pan, Q.H., Yanagisawa, Y., Sibayama, N., and Osa, T. (1994) The effects of chain-length of ferrocene moiety on electrical communication of mediators-modified and enzyme-modified electrodes. Denki Kagaku, 62 (12), 1240-1245. 

In a similar way to mediators, enzymes may be covalently bound to electrode surfaces, thus giving enzyme-chemically modified electrodes (ECME). When enzymes and mediators are coimmobilized, addition of auxiliary substances during the measuring process can be avoided a reagentless measuring regime becomes feasible (Fig. 19). 

The mediators are bound to amino acids near the prosthetic group. For fixation of the relays the protein has to be unfolded and renatured after the chemical modification procedure. The small distance between the bound mediator molecules (maximum 1 nm) provides a very fast tunneling process. Enzyme electrodes employing glucose oxidase or lactate oxidase modified in this way operate like mediator-modified electrodes, without reagent addition. Owing to their favorable structure, such sensors respond to the analyte in less than... 

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