Use of artificial mediators

Particularly useful have been the artificial mediators that shuttle electrons between the FAD center and the surface by the following scheme  

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USE OF ARTIFICIAL MEDIATOR ACCEPTING PYRIDINE NUCLEOTIDE OXIDOREDUCTASES (AMAPORs) FOR THE REGENERATION OF PYRIDINE NUCLEOTIDES... 

The concentration of hydrogen peroxide can be measured directly using amperometric detection. A change in H2O2 concentration in the medium appears as a variarion in the output current. The quantified parameters are m nitude of the sensor response, response time, and current response. It is desirable to measure signals in conditions when the linear relationship exists between the current value and the analyte concentration. At that point, the reactions are considered to be in steady state when pseudoequilibrium occurs between the species close to the sensor and their consumption at the indicative electrode. One of the serious problems associated with measurement of complex analytes is the possible interference of the redox species present in the sample. Several methods have been reported which aimed at reducing level of interference. These methods include use of perm-selective coatii, use of artificial mediators, or selective electrocatalysis. The use of mediators or selective electrocatalysis helps to lower the detection potential to the level when the majority of interferii species are electroinactive. ... 

Redox titrations or assays of active sites in complex enzymes may be carried out without use of artificial mediators. The advantage here of course is that the electron-carrier protein, as the natural mediator, is more likely to be site specific. Side reactions, such as non-enzymatic oxidation by dioxygen or peroxide (a frequent problem when using certain small-molecule mediators) are less likely to interfere. 

For the design of mitochondriotropic liposomes, we have used a method, that has been a standard procedure in liposome technology for over 30 years the lipid-mediated anchoring of artificially hydrophobized water-soluble molecules into liposomal membranes (25-28). We have hydrophobized mitochondriotropic TPP cations by conjugating them to long alkyl residues specifically, we have synthesized stearyl TPP (STPP) salts (29). Following liposome preparation in the presence of STPP, the liposomal surface became covalently modified with TPP cations, thereby rendering these liposomes mitochondriotropic as verified in vitro by fluorescence microscopy (30). 

A similar concept was used in the development of artificial chymotrypsin mimics [54]. The esterase-site was modeled by using the phosphonate template 75 as a stable transition state analogue (Scheme 13.19). The catalytic triad of the active site of chymotrypsin - that is, serine, histidine and aspartic acid (carboxy-late anion) - was mimicked by imidazole, phenolic hydroxy and carboxyl groups, respectively. The catalytically active MIP catalyst 76 was prepared using free radical polymerization, in the presence of the phosphonate template 75, methacrylic acid, ethylene glycol dimethacrylate and AIBN. The template removal conditions had a decisive influence on the efficiency of the polymer-mediated catalysis, and best results were obtained with aqueous Na2CC>3. 

Figure 6.5 Three generations of amperometric enzyme electrodes based on the use of natural secondary substrate (a), artificial redox mediators (b), or direct electron transfer between the enzyme and the electrode (c).

Clostridium thermoaceticum contains the so-called AMAPOR (artificial-media-tor-accepting pyridine-nucleotide oxidoreductases), which are useful for electro-microbial regeneration of all four forms of pyridine nucleotides, too. An NADP(H) dependent AMAPOR from C. thermoaceticum has been purified and characterized [104]. It is able to react with rather different artificial mediators such as viologens or quinones, for example 1,4-benzoquinone, anthraquinone-2,6-disulfonate, or 2,6-dichloro-indophenol. 

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