Enzyme microperoxidases

T. Lotzbeyer, W. Schuhmann, E. Katz, J. Falter, H.-L. Schmidt, Direct Electron Transfer between Covalently InunobUized Enzyme Microperoxidase MP-11 and a Cystamine-Modified Gold Electrode. J. Electroanal. Chem., 377 (1994) 291-294. 

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). 

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. 

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... 

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. 

Laszlo JA, Compton DL (2002) Comparison of peroxidase activities of hemin, cytochrome c and microperoxidase-11 in molecular solvents and imidazolium-based ionic liquids. J Mol Catal B Enzym 18 109-120... 

Nazari K, Mahmoudi A, Khosraneh M et al (2009) Kinetic analysis for suicide-substrate inactivation of microperoxidase-11 a modified model for bisubstrate enzymes in the presence of reversible inhibitors. J Mol Catal B Enzym 56 61-69... 

Several enzymes with peroxidase-like action have also been used in immunoassays. Microperoxidases are catalytically active fragments obtained from cytochrome c by proteolytic action. They consist of the heme group covalently coupled to a short peptide alpha helix (26). The active site structure is similar to that of peroxidase Four of the six possible coordination bonds of the iron atom are occupied by bonding to the porphyrin while the fifth complexes with a histidine residue and the sixth is exposed to the environment and forms the catalytically active portion of the molecule. The reaction mechanism and spectrum of substrates is similar to HRP, although the specific activity is variable... 

Figure 14. Scheme of the different steps involved in the fabrication of aligned shortened SWCNT arrays for direct electron transfer of enzymes such as microperoxidase MP-11. From reference 92. 

As in the case of horseradish peroxidase, several synthetic metalloporphyrins in the presence of H2O2 have been found to be potent catalysts for the chemiluminescent oxidation of luminol or isoluminol. The microperoxidases, mainly MP8 and MPll, have been shown to act as functional peroxidase enzyme models. " However, they are readily inactivated within one min of catalytic turnover, and incorporation into a molecular sieve... 

The most popular enzymes used in EIH are POase (Section 10.1.1), APase (Section 10.1.3), GOase (Section 10.1.4) and microperoxidase. 

Fig. 10.4. Microperoxidase (MPOase) obtained by proteolytic digestion of cytochrome c. Cleavage by pepsin yields a fragment of the active site of the enzyme containing 11 amino acids (11-MPOase), whereas trypsin yields a fragment of 9 amino acids (9-MPOase). Combination of these digestions yields 8-MPOase. These MPOases have a peroxidatic activity several hundred times greater than the intact cytochrome c.

Some effort has been devoted to obtain even smaller probes, i.e., smaller enzymes conjugated to Fab. An early candidate was cytochrome c (Singer, 1974) which has only 1/3 of the molecular weight of POase, though its diameter is only 30% smaller, but it has the disadvantage of low enzymatic activity. Digestion of cytochrome c (Section 10.3.1) yields the active site (microperoxidase, MPOase)... 

Microperoxidase-11, MP-11, is a heme-undecapeptide that is prepared by the digestion of cytochrome c and it includes the active surrounding of cytochrome MP-11 was immobilized on electrode surface s and its electrochemistry was characterized. The MP-11-modified electrodes were reported to act as effective electron mediator interfaces for the reduction of cytochrome c, hemoglobin, myoglobin and nitrate reductase (cytochrome c-dependent). The MP-11-mediated activation of nitrate reductase, NR, was employed to assemble an integrated MP-11/NR electrode for the bioelectrocatalyzed reduction of nitrate (NO3 ) to nitrite (NO2). An affinity complex between a MP-11-functionalized electrode and NR (/Q = 3.7x10 M ) was crosslinked with glutaric dialdehyde to yield the electrically contacted electrode for the bioelectrocatalyzed reduction of nitrate to nitrite. In this system the reduced MP-11 mediates ET to NR and activates the enzyme towards the reduction of NO3. 

The oxidation of iodide is performed by enzymes of the peroxidase type (Morrison, 1980), mainly lactoperoxidase, but also by horseradish peroxidase, microperoxidase and chloroperoxidase, which are hemoproteins with tetrapyr-rolic cores chelating iron. These enzymes are active in the presence of small amounts of hydrogen peroxide or glucose oxidase. They form a first complex with H2O2 that... 

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