Microperoxidases

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

Fig. 12.2. Free energy data for electron transfer between the protein cytochrome c and the small acceptor microperoxidase-8 (MP8), from recent simulations [47]. Top Gibbs free energy derivative versus the coupling parameter A. The data correspond to solvated cytochrome c the MP8 contribution is not shown (adapted from [47]) Bottom the Marcus diabatic free energy curves. The simulation data correspond to cyt c and MP8, infinitely separated in aqueous solution. The curves intersect at 77 = 0, as they should. The reaction free energy is decomposed into a static and relaxation component, using the two steps shown by arrows a static, vertical step, then relaxation into the product state. All free energies in kcalmol-1. Adapted with permission from reference [88]...

Ichinose, H. Wariishi, H., and Tanaka, H., Effective oxygen transfer reaction catalyzed by microperoxidase-11 during sulfur oxidation of dibenzothiophene. Enzyme and Microbial Technology, 2002. 30 pp. 334—339. 

Recently, direct electron transfer to microperoxidases adsorbed on carbon nanotube-modified platinum electrodes has been observed [24], The redox potential for this direct electron transfer is-0.4 V vs SCE, the same as that for the microper-... 

Moreover, it has been demonstrated that CNTs promote the direct electrochemistry of enzymes. Dong and coworkers have reported the direct electrochemistry of microperoxidase 11 (MP-11) using CNT-modified GC electrodes [101] and layer-by-layer self-assembled films of chitosan and CNTs [102], The immobilized MP-11 has retained its bioelectrocatalytic activity for the reduction of H202 and 02, which can be used in biosensors or biofuel cells. The direct electrochemistry of catalase at the CNT-modified gold and GC electrodes has also been reported [103-104], The electron transfer rate involving the heme Fe(III)/Fe(II) redox couple for catalase on the CNT-modified electrode is much faster than that on an unmodified electrode or other... 

M. Wang, Y. Shen, Y. Liu, T. Wang, F. Zhao, B. Liu, and S. Dong, Direct electrochemistry of microperoxidase 11 using carbon nanotube modified electrodes. J. Electroanal. Chem. 578, 121-127 (2005). 

Z. Xu, N. Gao, H. Chen, and S. Dong, Biopolymer and carbon nanotubes interface prepared by self-assembly for studying the electrochemistry of microperoxidase-11. Langmuir 21, 10808-10813 (2005). 

F. Patolsky, T. Gabriel, and I. Willner, Controlled electrocatalysis by microperoxidase-11 and Au-nano-particle superstructures on conductive supports. J. Electroanal. Chem. 479, 69-73 (1999). 

Amino acids Microperoxidase-catalyzed Isoluminol- H202 Borate buffer (pH 9.3)... 

Luminol Microperoxidase-catalyzed ILITC- H202 Phosphate buffer (pH 10.8) Compact detection cell using optical fiber 14 amol (for luminol) 110... 

CL reaction can be catalyzed by enzymes other than HRP (e.g., microperoxidase and catalase) and by other substances [hemoglobin, cytochrome c, Fe(III), and other metal complexes]. The presence of suitable molecules such as phenols (p-iodophenol), naphthols (l-bromo-2-naphthol), or amines (p-anisidine) increases the light production deriving from the HRP-catalyzed oxidation of luminol and produces glow-type kinetics [6, 7], The use of other enzymes, such as glucose-6-phosphate dehydrogenase [38-41], P-galactosidase [42], and xanthine oxidase [43-46], as CL labels has been reported. 

Peroxynitrite reacts with heme proteins such as prostacycline synthase (PGI2), microperoxidase, and the heme thiolate protein P450 to form a ferryl nitrogen dioxide complex as an intermediate [120]. Peroxynitrite also reacts with acetaldehyde with the rate constant of 680 1 mol 1 s" 1 forming a hypothetical adduct, which is decomposed into acetate, formate, and methyl radicals [121]. The oxidation of NADH and NADPH by peroxynitrite most certainly occurs by free radical mechanism [122,123], Kirsch and de Groot [122] concluded that peroxynitrite oxidized NADH by a one-electron transfer mechanism to form NAD and superoxide ... 

Tanaka T (1984) Transport pathway and uptake of microperoxidase in the junctional epithelium of healthy rat gingiva. J Periodontal Res 19 26-39... 

The main idea demonstrated by Willner and coworkers [20] is the ability to construct multilayered nanoparticle electrodes, which are porous. In a related study Patolsky et al. extended this idea further using biocatalysts to detect H2O2 [18]. In this example, the construction of the electrode is similar to the one described above but the redox-active bridging molecule was replaced with microperoxidase-11 (MP-11). 

Figure 1.33 Magneto-controlled on ofP switching of microperoxidase-11-catalyzed reduction of cumene hydroperoxide in an organic environment. (Reprinted with permission from Ref [166]. 2000 American Chemical Society.)...

Direct electron transfer has also been achieved with many metalloproteins such as cytochrome C, horseradish peroxidase, microperoxidase (MP-11), myoglobin, hemoglobin, catalase, azurin, and so on, immobilized on different CNT-modified electrodes [45, 61, 144—153]. 

Fatty acid hydroperoxides can be separated from each other and other lipids by MEKC followed by FLD according to equation 34 with 106a, using as catalyst microperoxidase-11 immobilized on the wall of a small capillary coupled at the end of the electrophoresis track. MEKC with DA-UVD can be applied for separation of unsaturated fatty acids from the mixture of hydroperoxides obtained on oxidation with 102 . ... 

The CLD methods for HPLC using isoluminol (190) with microperoxidase catalysis, for determination of lipid hydroperoxides in clinical fluids, have been reviewed. Determination of phospholipids hydroperoxides by luminol (124) CL has been reviewed . A fast RP-HPLC method (retention times 1 to 2 min) for determination of hydroperoxides and other peroxide compounds includes UVD, which is not always effective, and CLD, attained on injection of luminol (124), the CL reagent (Scheme 3), hemin (75a), a catalyst, and NaOH to raise the pH of the solution. A FLD cell may act as CLD cell if the excitation source is turned off. The selectivity of CLD is of advantage over UVD in industrial analysis thus, for example, UVD of a sample from a phenol production line based on cumene oxidation (equation 13) shows peaks for cumyl hydroperoxide (27), unreacted cumene, cumyl alcohol and acetophenone, whereas CLD shows only the 27 peak. The... 

A procedure for determination of lipid hydroperoxides in human plasma is based on kinetic measurement of the CL of luminol (124) with hemin (75a) catalysis . CLD of microperoxidase-catalyzed oxidation of luminol (124) or isoluminol (190) was applied to detection and determination of amino acid hydroperoxides after exposure to UV and y-irradiation A method for determination of hydroperoxides in the phospholipids of cultured cells uses isoluminol (190) and microperoxidase as catalyst " . Simultaneous determination of phosphatidylcholine hydroperoxides and cholesteryl ester hydroperoxides in human serum is carried out by quantitative extraction of the lipids, HPLC separation by column switching and CLD using isoluminol (190) with microperoxidase catalysis . ... 

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