Horseradish peroxidase enzymatic activity

Answers to these questions were initiated over a decade ago during our studies on catalase (CAT) and horseradish peroxidase (HRP) (30). Both native enzymes are ferric hemoproteins and both are oxidized by hydrogen peroxide. These oxidations cause the loss of two electrons and generate active enzymatic intermediates that can be formally considered as Fe + complexes. 

LiP catalyzes the oxidation of a low-molecular-weight redox mediator, veratryl alcohol, which in mrn mediates one-electron oxidation of lignin to generate aryl cation radicals [100]. The radicals facilitate a wide variety of reactions such as carbon-carbon cleavage, hydroxylation, demethylation, and so on. Dezotti et al. [101] reported enzymatic removal of color from extraction stage effluents using lignin and horseradish peroxidases immobilized on an activated silica gel support. 

This problem was solved by Adam and coworkers in 1994-1998. They presented a high-yielding and diastereoselective method for the preparation of epoxydiols starting from enantiomerically pure allyhc alcohols 39 (Scheme 69). Photooxygenation of the latter produces unsaturated a-hydroxyhydroperoxides 146 via Schenck ene reaction. In this reaction the (Z)-allylic alcohols afford the (5, 5 )-hydroperoxy alcohols 146 as the main diastereomer in a high threo selectivity (dr >92 8) as racemic mixmre. The ( )-allylic alcohols react totally unselectively threolerythro 1/1). Subsequent enzymatic kinetic resolution of rac-146 threolerythro mixture) with horseradish peroxidase (HRP) led to optically active hydroperoxy alcohols S,S) and (//,5 )-146 ee >99%) and the... 

Besides using the bioactive agent to detect the ion of interest, another approach can include monitoring an ion by its inhibitory effect upon enzymatic activity. For example, horseradish peroxidase (HRP) can be immobilised onto one gate of a REFET [107] allowing the presence of cyanide ion to be measured at concentrations of 10 3-10 7 M. The approach used here is to monitor the inhibition of the enzymatic HRP effect, by the cyanide ion, on ascorbic acid. Even lower levels (10 10 M) of detection can be obtained using a polyphenol oxidase/clay composite immobilised on carbon, with no interference from chloride, nitrate or bromide [108]. 

Adsorbed horseradish peroxidase (HRP) on silica mesoporous materials The immobilized HRP on FSM-16 and MCM-41 with pore diameter of 50 A showed the highest enzymatic activity in toluene and thermostability in aqueous solution at a temperature of 70°C [4]... 

Wang Z, King TL, Branagan SP et al (2009) Enzymatic activity of surface-immobilized horseradish peroxidase confined to micrometer-to nanometer-scale structures in nanocapillary array membranes. Analyst 134 851-859... 

Nielsen KL, Indiani C, Henriksen A et al (2001) Differential activity and structure of highly similar peroxidases. Spectroscopic, crystallographic, and enzymatic analyses of lignifying Arabidopsis thaliana peroxidase A2 and horseradish peroxidase A2. Biochemistry 40 1103-11021... 

Horseradish peroxidase is an excellent candidate with which to elucidate enzymatic kinetics in organic solvents. It is an active enzyme with turnover numbers exceeding 320 s 1 in organic media (XI) and hence is susceptible to diffusional limitations which must be overcome. Peroxidase also catalyzes mechanistically identical reactions in aqueous and organic media. Therefore, direct kinetic comparisons between aqueous and organic reactions can be made and the effects of the organic solvent on reactivity and substrate specificity can be directly compared to aqueous-based catalysis. 

The insolubility of enzymes in monophasic organic systems has a controlling influence on the kinetics of enzymatic catalysis in organic media. Insolubilized enzymes are subject to intraparticle and external diffusional limitations which can mask the true, intrinsic kinetics of catalysis. These limitations are particularly severe for highly active and purified enzymes such as horseradish peroxidase. One way to overcome this problem is to increase the surface area of the enzyme in contact with the organic solvent. 

The flexibility of the sol-gel process allows multiple approaches to enzymatic activity monitoring. The formation of hydrogen peroxide can be followed by optical measmements using another enzymatic reaction in which the oxidation of an organic dye is catalyzed by a peroxidase (HorseRadish Peroxidase HRP), coentrapped with GOD. The presence of glucose in the solution can also be detected via electrochemical means by following the redox reactions at the active site of GOD. However, because of the steric hindrance of the protein molecule, a ferrocene mediator has to be used in order to transfer electrons from the hidden active site to the electrode. Alternatively, oxygen consumption can be measmed with a Clark electrode. The hybrid gel is deposited on a Pt cathode... 

Suleiman and Xu [128] described a novel reusable amperometric immuno-sensor for the determination of cocaine. Horseradish peroxidase and benzoylecgonine-antibody were co-immobilized on a chemically activated affinity membrane, which was then mounted over the tip of an oxygen electrode. The enzymatic electrocatalytic current response to the substrate is inhibited by the association of the antigen to the co-immobilized antibody. The calibration plot for cocaine was linear in the concentration range of lx 10 -1x10 M. 

Recently, some papers have started to highlight the performance of PSf-(bio) composites-CNT as electrode material for electrochemical immunosensing [112]. The authors have highlighted the attractive combination of PSf/CNTplus disposable screen-printed electrodes for monitoring the enzymatic activity of horseradish peroxidase and the RIgG inmunosensor response. In both cases an enhanced electroanalytical response was demonstrated in comparison with standard graph-ite/PSf composites. 

Such ILCE showed remarkably high enantioselectivity for transesterification reaction of several secondary alcohols in the presence of vinyl acetate in toluene, and there was no significant deterioration in activity even after multiple (five times) use. Horseradish peroxidase (HRP) is another example to be coated with both hydrophobic and hydrophilic ILs such as [BMIMJITf N] and [BMIM][PEg] toward enzymatic synthesis of polyaniline [23]. An attractive point of the ILCE-based immobilization method is easy recovery of enzyme/IL phase by liquid-liquid phase separation and effective reusability of the enzyme (Eigs. 10.8, 10.9) [23, 59]. 

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