Activity Of Horseradish Peroxidase Research Articles

Immobilization of horseradish peroxidase by electrospun fibrous membranes for adsorption and degradation of pentachlorophenol in water

Horseradish peroxidase (HRP) is successfully in situ encapsulated into the poly(d,l-lactide-co-glycolide) (PLGA)/PEO–PPO–PEO (F108) electrospun fibrous membranes (EFMs) by emulsion electrospinning. The adsorption and degradation of pentachlorophenol (PCP) by HRP–EFMs are investigated. The experimental results show that the sorption kinetic of PCP on EFMs follows the pseudo-second-order model, and the sorption capacity is as high as 44.69mgg−1. The sorption mechanisms of EFMs for PCP can be explained by hydrogen bonding interactions, hydrophobic interactions and π–π bonding interactions. Profiting from the strong adsorption, the removal of PCP can be dramatically enhanced by the interaction of adsorbed PCP and HRP on the surface of EFMs. For PCP degradation, the optimal pH values for free HRP and immobilized HRP are 4 and 2–4, respectively. As pH>4.7, no adsorption and degradation are observed due to the deprotonation of PCP. The removal percentages reach 83% and 47% for immobilized HRP and free HRP, respectively, at 25±1°C. The presence of humic acid can inhibit the activity of HRP and decreases the adsorption capacity of PCP because of competitive adsorption. The operational and storage stability of immobilized HRP are highly improved through emulsion electrospinning.

In situ synthesized gold nanoparticles for direct electrochemistry of horseradish peroxidase

In order to construct a three-dimensional electrode, in situ electrochemical deposition of gold nanoparticles onto a gold electrode coated with a self-assembled monolayer of 3-mercaptopropionic acid (Au NP/MPA/Au) was conducted. Horseradish peroxidase (HRP) was then immobilized into this three-dimensional electrode, leading to the realization of direct electron transfer of HRP. Scanning electron microscopy and electrochemical impedance spectroscopy were applied to characterize the electrode before and after HRP immobilization. The apparent Michaelis-Menten constant for immobilized HRP on a Au NP/MPA/Au electrode is 0.78mM, indicating high enzymatic activity of HRP. The HRP modified electrode was further utilized as a sensing platform for the electrocatalytic reduction and detection of hydrogen peroxide. For the detection of hydrogen peroxide on this electrode, the sensitivity is 311.72μAmM−1cm−2, the detection limit is 0.16μM, and the dynamic range is from 0.48μM to 1.2mM. Therefore this electrode is a promising device for sensitive and reproducible detection of H2O2.

Water-soluble electrospun nanofibers as a method for on-chip reagent storage.

This work demonstrates the ability to electrospin reagents into water-soluble nanofibers resulting in a stable on-chip enzyme storage format. Polyvinylpyrrolidone (PVP) nanofibers were spun with incorporation of the enzyme horseradish peroxidase (HRP). Scanning electron microscopy (SEM) of the spun nanofibers was used to confirm the non-woven structure which had an average diameter of 155 ± 34 nm. The HRP containing fibers were tested for their change in activity following electrospinning and during storage. A colorimetric assay was used to characterize the activity of HRP by reaction with the nanofiber mats in a microtiter plate and monitoring the change in absorption over time. Immediately following electrospinning, the activity peak for the HRP decreased by approximately 20%. After a storage study over 280 days, 40% of the activity remained. In addition to activity, the fibers were observed to solubilize in the microfluidic chamber. The chromogenic 3,3′,5,5′-tetramethylbenzidine solution reacted immediately with the fibers as they passed through a microfluidic channel. The ability to store enzymes and other reagents on-chip in a rapidly dispersible format could reduce the assay steps required of an operator to perform.

Evaluation of a horseradish peroxidase‐catalyzed process for triclosan removal and antibacterial activity reduction

AbstractBACKGROUND: Triclosan is a biocide used in personal care products, and its presence in water bodies has been reported. This compound is suspected to be linked with the emergence of antibiotic‐resistant bacteria. In the present work, the enzyme horseradish peroxidase (HRP) was used to catalyze triclosan removal.RESULTS: The stoichiometric ratio of H2O2 to triclosan (0.83) was higher than the value predicted by the HRP catalytic cycle (0.5). During the reactions, HRP activity was gradually reduced, likely due to excess H2O2. The addition of veratryl alcohol, syringaldazine and p‐coumaric acid (redox mediators) to the reaction medium, at a mediator/triclosan molar ratio of 1.0, increased triclosan removal from 45% (without a mediator) to 56%, 64% and 80%, respectively. The antibacterial activity of these mediators was much lower than that observed for triclosan.CONCLUSION: The antibacterial activity of triclosan solution was effectively reduced after enzymatic treatment. Moreover, the enzymatic process was proven to be technically feasible for removing triclosan at an environmental relevant concentration. © 2012 Society of Chemical Industry

A Bioelectrochemical Method for the Quantitative Description of the Hofmeister Effect of Ionic Liquids in Aqueous Solution

It is imperative to establish a simple, efficient, and practical method to investigate the Hofmeister effect of ionic liquids (ILs) on the behavior of proteins (enzymes). In this study, the effects of the cations and anions of different ILs in aqueous media on the structural stability of horseradish peroxidase (HRP), a model oxidoreductase, were systematically investigated using electrochemical methods. It is found that without ILs no direct electron transfer current signals of HRP appear at bare glassy carbon electrode (GCE) in phosphate buffer (pH 7.0) even after incubation and accumulation at a negative potential. In the presence of ILs, however, a current signal occurs at GCE, depending on the structure of the IL and its concentration. A linear relationship between the peak currents and the scan rates demonstrates that the direct electron transfer is a surface-confined thin-layer electrochemical process. The redox signal at GCE is from the heme of HRP. An IL has a perturbing effect on the HRP structure. The anodic peak current of HRP at GCE, the catalytic activity of HRP, and the secondary structure of HRP are well correlated. Different cations or anions at varied concentrations have different effects on the structural stability of HRP, resulting in different current signals at GCE. Thus, the anodic peak current of HRP at GCE can be used as an indicator to quantitatively characterize the effect of ILs on the structural stability of HRP. The present Hofmeister series for cations and anions is in good agreement with that reported elsewhere. To our knowledge, this is a first attempt to establish a simple and practical electrochemical method to correlate Hofmeister effects with characteristics of ions and solvents. The present investigation not only deepens our understanding of the complex electrochemical behavior of proteins in ILs media but also offers a practical guidance to designing "green" and biocompatible ILs for protein (enzyme) separation, purification, and enzymatic catalysis and conversion.

Development of a quantitative diagnostic method of estrogen receptor expression levels by immunohistochemistry using organic fluorescent material-assembled nanoparticles

The detection of estrogen receptors (ERs) by immunohistochemistry (IHC) using 3,3′-diaminobenzidine (DAB) is slightly weak as a prognostic marker, but it is essential to the application of endocrine therapy, such as antiestrogen tamoxifen-based therapy. IHC using DAB is a poor quantitative method because horseradish peroxidase (HRP) activity depends on reaction time, temperature and substrate concentration. However, IHC using fluorescent material provides an effective method to quantitatively use IHC because the signal intensity is proportional to the intensity of the photon excitation energy. However, the high level of autofluorescence has impeded the development of quantitative IHC using fluorescence. We developed organic fluorescent material (tetramethylrhodamine)-assembled nanoparticles for IHC. Tissue autofluorescence is comparable to the fluorescence intensity of quantum dots, which are the most representative fluorescent nanoparticles. The fluorescent intensity of our novel nanoparticles was 10.2-fold greater than quantum dots, and they did not bind non-specifically to breast cancer tissues due to the polyethylene glycol chain that coated their surfaces. Therefore, the fluorescent intensity of our nanoparticles significantly exceeded autofluorescence, which produced a significantly higher signal-to-noise ratio on IHC-imaged cancer tissues than previous methods. Moreover, immunostaining data from our nanoparticle fluorescent IHC and IHC with DAB were compared in the same region of adjacent tissues sections to quantitatively examine the two methods. The results demonstrated that our nanoparticle staining analyzed a wide range of ER expression levels with higher accuracy and quantitative sensitivity than DAB staining. This enhancement in the diagnostic accuracy and sensitivity for ERs using our immunostaining method will improve the prediction of responses to therapies that target ERs and progesterone receptors that are induced by a downstream ER signal.

Enzymatic Growth of Quantum Dots: Applications to Probe Glucose Oxidase and Horseradish Peroxidase and Sense Glucose

Three innovative assays are developed for the detection of enzymatic activities of glucose oxidase (GOx) and horseradish peroxidase (HRP) by the generation of CdS quantum dots (QDs) in situ using non-conventional enzymatic reactions. In the first assay, GOx catalyzes the oxidation of 1-thio-β-D-glucose to give 1-thio-β-D-gluconic acid. The latter is spontaneously hydrolyzed to β-D-gluconic acid and H2 S, which in the presence of cadmium nitrate yields fluorescent CdS nanoparticles. In the second assay HRP catalyzes the oxidation of sodium thiosulfate with hydrogen peroxide generating H2 S and consequently CdS QDs. The combination of GOx with HRP, allowed quantification of glucose in plasma by following growth of fluorescent QDs.

DNA-directed immobilization of horseradish peroxidase onto porous SiO2 optical transducers

Multifunctional porous Si nanostructure is designed to optically monitor enzymatic activity of horseradish peroxidase. First, an oxidized PSi optical nanostructure, a Fabry-Pérot thin film, is synthesized and is used as the optical transducer element. Immobilization of the enzyme onto the nanostructure is performed through DNA-directed immobilization. Preliminary studies demonstrate high enzymatic activity levels of the immobilized horseradish peroxidase, while maintaining its specificity. The catalytic activity of the enzymes immobilized within the porous nanostructure is monitored in real time by reflective interferometric Fourier transform spectroscopy. We show that we can easily regenerate the surface for consecutive biosensing analysis by mild dehybridization conditions.

HMGN1 Protein Regulates Poly(ADP-ribose) Polymerase-1 (PARP-1) Self-PARylation in Mouse Fibroblasts

In mammalian cells, the nucleosome-binding protein HMGN1 (high mobility group N1) affects the structure and function of chromatin and plays a role in repair of damaged DNA. HMGN1 affects the interaction of DNA repair factors with chromatin and their access to damaged DNA; however, not all of the repair factors affected have been identified. Here, we report that HMGN1 affects the self-poly(ADP-ribosyl)ation (i.e., PARylation) of poly(ADP-ribose) polymerase-1 (PARP-1), a multifunctional and abundant nuclear enzyme known to recognize DNA lesions and promote chromatin remodeling, DNA repair, and other nucleic acid transactions. The catalytic activity of PARP-1 is activated by DNA with a strand break, and this results in self-PARylation and PARylation of other chromatin proteins. Using cells obtained from Hmgn1(-/-) and Hmgn1(+/+) littermate mice, we find that in untreated cells, loss of HMGN1 protein reduces PARP-1 self-PARylation. A similar result was obtained after MMS treatment of these cells. In imaging experiments after low energy laser-induced DNA damage, less PARylation at lesion sites was observed in Hmgn1(-/-) than in Hmgn1(+/+) cells. The HMGN1 regulation of PARP-1 activity could be mediated by direct protein-protein interaction as HMGN1 and PARP-1 were found to interact in binding assays. Purified HMGN1 was able to stimulate self-PARylation of purified PARP-1, and in experiments with cell extracts, self-PARylation was greater in Hmgn1(+/+) than in Hmgn1(-/-) extract. The results suggest a regulatory role for HMGN1 in PARP-1 activation.

Low concentration of silver nanoparticles not only enhances the activity of horseradish peroxidase but alter the structure also.

Chemical synthesis of Ag-NPs was carried out using reduction method. The reduction mechanistic approach of silver ions was found to be a basic clue for the formation of the Ag-NPs. The nanoparticles were characterized by UV-vis, FT-IR and TEM analysis. We had designed some experiments in support of our hypothesis, “low concentrations of novel nanoparticles (silver and gold) increases the activity of plant peroxidases and alter their structure also”, we had used Ag-NPs and HRP as models. The immobilization/interaction experiment had demonstrated the specific concentration range of the Ag-NPs and within this range, an increase in HRP activity was reported. At 0.08 mM concentration of Ag-NPs, 50% increase in the activity yield was found. The U.V-vis spectra had demonstrated the increase in the absorbance of HRP within the reported concentration range (0.06–0.12 mM). Above and below this concentration range there was a decrease in the activity of HRP. The results that we had found from the fluorescence spectra were also in favor of our hypothesis. There was a maximum increase in ellipticity and α-helix contents in the presence of 0.08 mM concentration of Ag-NPs, demonstrated by circular dichroism (CD) spectra. Finally, incubation of a plant peroxidase, HRP with Ag-NPs, within the reported concentration range not only enhances the activity but also alter the structure.

Supported biocatalysts for Alizarin and Eriochrome Blue Black R degradation using hydrogen peroxide

The goal of this manuscript was to study the immobilization of horseradish peroxidase (HRP) and hematin on different supports at selected conditions to be used in the removal of two synthetic dyes: Alizarin Red (AR) and Eriochrome Blue Black R (EBBR). Reuse in the target reactions was tested for the best biocatalysts. Three supports were selected: magnetite-activated carbon Fe3O4/C, activated carbon C and chitosan CS. Several reasons led to discard the use of Fe3O4/C and C as supports for hematin and HRP. CS–glutaraldehyde (GA)–HRP not only has low ability to remove EBBR, but also the enzymatic activity of HRP could not be separated from the dye adsorption on CS. However, some adsorptive functions of CS–GA were lost when HRP was supported on it.In the case of CS–GA–hematin, the most important factors for EBBR removal are dye concentration and mass of catalyst, whereas the temperature has almost no effect on the conversion of dye, considering in all cases the H2O2 concentration (0.021M) constant.There was not a marked decrease in the removal of AR even after the ninth reuse in the case of CS–GA–hematin. For EBBR removal, with CS–GA–HRP, in the second reuse the catalyst was completely deactivated, whereas with CS–GA–hematin this was found only in the fourth reuse.

One-step electrochemical immunosensing for simultaneous detection of two biomarkers using thionine and ferrocene as distinguishable signal tags

We report on a new kind of electrochemical immunosensors for simultaneous determination of the biomarkers carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP). Thionine and ferrocene were applied as distinguishable electrochemical tags (and mediators) which were covalently conjugated on anti-AFP and anti-CEA antibodies, respectively, via carboxy groups. The resulting conjugates were co-immobilized on a glassy carbon electrode functionalized with gold nanoparticles. Finally, horseradish peroxidase (HRP) was immobilized onto the modified electrode. Labeled thionine and ferrocene, respectively, act as distinguishable tags for simultaneous determination of AFP and CEA due to the difference in the location of their voltammetric peaks. With a one-step immunoassay format, the analytes in the sample produced transparent immunoaffinity reaction with the corresponding antibodies on the electrode. Once the immunocomplex is formed, it partially inhibits the active center of the immobilized HRP, and this decreased the activity of HRP in terms of reduction of hydrogen peroxide. This immunosensor enables the simultaneous determination of AFP and CEA in a single run and within the same dynamic range (0.01–50 ng mL−1) and the same lower detection limit (0.01 ng mL−1). The reproducibility and stability of the immunosensors are acceptable. The dual immunosensor was applied to evaluate several specimens, and the assay results are in acceptable agreement with clinical data.

Effect of organic solvents on peroxidases from rice and horseradish: Prospects for enzyme based applications

A feasibility test for rice peroxidase (RP) enzyme as a substitute for horseradish peroxidase (HRP) was carried out. The activity of HRP was maximum at 30°C with pH 6.0–7.0. The purified rice peroxidase showed optimum activity at 30°C with pH 7–8 and was thermostable till 68°C, which is higher than the temperature reported for HRP. RP obeyed Michaelis–Menten kinetics. With increasing substrate concentrations, RP and HRP had Vmax as 8.23μMmin−1 and 4.21μMmin−1 and Km as 5.585 and 3.662mM, respectively. In 10% 1,4-dioxane and ethanol, RP exhibited 2 and 1.3 times higher activity, respectively than HRP. Shelf life studies show RP to be significantly stable till 60h in 20% 1,4-dioxane and till 12h in ethanol. The activity of RP/HRP increased gradually with 0%–40% ethanol or 0%–30% 1,4-dioxane till 20h with a sharp decline thereafter. The stability of HRP and RP reduced with increasing storage period. Enzyme efficiencies compared as Vm/Km showed water miscible organic solvents, viz.1,4-dioxane and ethanol, to exhibit a regular decrease in Vm/Km with increase in organic solvent concentration whereas, a reverse trend was observed with water–immiscible solvent like chloroform. The relative activity of RP and HRP enzymes upon immobilization on poly-5-carboxy-indole shows increasing enzyme activity with time and with guaiacol/dopamine hydrochloride as substrates. Immobilized RP had a better relative activity with dopamine as substrate than immobilized HRP, whereas with guaiacol both RP and HRP had a comparable activity upon immobilization. Results suggest rice peroxidase to be a cheaper and convenient enzyme system for immobilization using organic solvents. The high thermal stability, more stability in organic solvents and longer shelf life of RP over the immobilizing matrix suggest conducting polyindole having carboxyl functional groups to be a suitable matrix for the covalent entrapment of rice peroxidase through amide linkage. Good sensitivity and fast response to dopamine makes RP a suitable enzyme system for monitoring dopamine levels in aqueous medium.

Enzyme activity assay for horseradish peroxidase encapsulated in peptide nanotubes

Encapsulation of horseradish peroxidase (HRP) inside a peptide nanotube (PNT) was demonstrated and its activity was measured. Enzyme assay verified that 0.16μg of the enzymes were encapsulated in 1mg of PNTs. The encapsulation was also verified with TEM, UV–vis spectroscopy, and FTIR. The activity of the encapsulated HRP was examined for thermal stability, long-term storage stability, and resistance to a denaturant. They showed good storage stability, retaining its activity up to 90%, while the free HRP lost 50% of its activity over the course of 18days. At 55°C, the encapsulated HRP activity remained 20% higher than that of the free HRP. With the denaturant, guanidinium hydrochloride (GdmHCl), the encapsulated HRP activity was maintained around 10% higher than the free HRP. This result proves that the encapsulation of HRP inside the PNT may be an effective way to keep the enzyme activity stable in various environments.

Enhanced stability of horseradish peroxidase encapsulated in acetalated dextran microparticles stored outside cold chain conditions

Micro- and nanoparticles have been shown to improve the efficacy of safer protein-based (subunit) vaccines. Here, we evaluate a method of improving the vaccine stability outside cold chain conditions by encapsulation of a model enzyme, horseradish peroxidase (HRP), in an acid-sensitive, tunable biodegradable polymer, acetalated dextran (Ac-DEX). Vaccines that are stable outside the cold chain would be desirable for use in developing nations. Ac-DEX particles encapsulating HRP were prepared using two different methods, probe sonication and homogenization. These particles were stored under different storage conditions (−20°C, 4°C, 25°C or 45°C) for a period of 3 months. On different days, the particles were characterized for various physical and chemical measurements. At all conditions, Ac-DEX particles remained spherical in nature, as compared to PLGA particles that fused together starting at day 3 at 45°C. Furthermore, our results indicated that encapsulation of HRP in Ac-DEX reduces its storage temperature dependence and enhances its stability outside cold chain conditions. Homogenized particles performed better than probe sonicated particles and retained 70% of the enzyme's initial activity as compared to free HRP that retained only 40% of the initial activity after 3 months of storage at 25°C or 45°C. Additionally, HRP activity was more stable when encapsulated in Ac-DEX, and the variance in enzyme activity between the different storage temperatures was not observed for either particle preparation. This suggests that storage at a constant temperature is not required with vaccines encapsulated in Ac-DEX particles. Overall, our results suggest that an Ac-DEX based micro-/nanoparticles system has wide applications as vaccines and drug delivery carriers, including those in developing nations.

Strategies for an enzyme immobilization on electrodes: Structural and electrochemical characterizations

In this paper, we propose various strategies for an enzyme immobilization on electrodes (both metal and semiconductor electrodes). In general, the proposed methodology involves two critical steps viz., (1) chemical modification of substrates using functional monolayers [Langmuir – Blodgett (LB) films and/or self-assembled monolayers (SAMs)] and (2) anchoring of a target enzyme using specific chemical and physical interactions by attacking the terminal functionality of the modified films. Basically there are three ways to immobilize an enzyme on chemically modified electrodes. First method consists of an electrostatic interaction between the enzyme and terminal functional groups present within the chemically modified films. Second and third methods involve the introduction of nanomaterials followed by an enzyme immobilization using both the physical and chemical adsorption processes. As a proof of principle, in this work we demonstrate the sensing and catalytic activity of horseradish peroxidase (HRP) anchored onto SAM modified indium tin oxide (ITO) electrodes towards hydrogen peroxide (H2O2). Structural characterization of such modified electrodes is performed using X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and contact angle measurements. The binding events and the enzymatic reactions are monitored using electrochemical techniques mainly cyclic voltammetry (CV).

Promoting immobilization and catalytic activity of horseradish peroxidase on mesoporous silica through template micelles

New concept on the promotion of immobilization and catalytic activity of enzyme on mesoporous silica through template micelles is proposed and realized in this paper. Proper P123 templates are controllable retained in the as-synthesized SBA-15, not only to anchor the horseradish peroxidase (HRP) guest, but also to establish the crowding-like microenvironment around the enzyme. The influence of retaining templates on the pore structure of SBA-15, immobilization, and catalytic activity of HRP is studied, and the possible process of template removal is proposed. Ethanol refluxing of 6h is conformable to prepare the optimal mesoporous support characterized with the retained templates of about 8%. With the assistance of retained templates in SBA-15, up to 49mgg−1 of HRP can be immobilized, 100% more than that on calcined SBA-15. Furthermore, the thermal stability, the resistance of pH variation and denaturing agent urea, and the recycle usage of HRP immobilized are obviously elevated, paving a novel and low-cost route to develop enzyme catalysts.

Enzyme‐Regulated Activation of DNAzyme: A Novel Strategy for a Label‐Free Colorimetric DNA Ligase Assay and Ligase‐Based Biosensing

The DNA nick repair catalyzed by DNA ligase is significant for fundamental life processes, such as the replication, repair, and recombination of nucleic acids. Here, we have employed ligase to regulate DNAzyme activity and developed a homogeneous, colorimetric, label-free and DNAzyme-based strategy to detect DNA ligase activity. This novel strategy relies on the ligation-trigged activation or production of horseradish peroxidase mimicking DNAzyme that catalyzes the generation of a color change signal; this results in a colorimetric assay of DNA ligase activity. Using T4 DNA ligase as a model, we have proposed two approaches to demonstrate the validity of the DNAzyme strategy. The first approach utilizes an allosteric hairpin-DNAzyme probe specifically responsive to DNA ligation; this approach has a wide detection range from 0.2 to 40 U mL(-1) and a detection limit of 0.2 U mL(-1). Furthermore, the approach was adapted to probe nucleic acid phosphorylation and single nucleotide mismatch. The second approach employs a "split DNA machine" to produce numerous DNAzymes after being reassembled by DNA ligase; this greatly enhances the detection sensitivity by a signal amplification cascade to achieve a detection limit of 0.01 U mL(-1).

Design and Preparation of a Nanoprobe for Imaging Inflammation Sites

To image inflammation sites, we developed a novel nanoparticle, hydroxylamine-containing nanoparticle (HANP), which emits an intense electron spin resonance (ESR)-signal triggered by enzymatic oxidation reaction and pH-sensitive self-disintegration. The nanoparticle was prepared from an amphiphilic block copolymer, poly(ethylene glycol)-b-poly[4-(2,2,6,6-tetramethylpiperidine-1-hydroxyl)aminomethylstyrene] (PEG-b-PMNT-H), which spontaneously forms a core-shell type polymeric micelle (particle diameter = ca. 50 nm) in aqueous media. Because the PMNT-H segment in the block copolymer possesses amino groups in each repeating unit, the particle can be disintegrated by protonation of the amino groups in an acidic pH environment such as inflammation sites, which is confined to the hydrophobic core of HANP. Mixing HANP with horseradish peroxidase (HRP)/H(2)O(2) mixture resulted in enzymatic oxidization of the hydroxylamines in the PEG-b-PMNT-H and converted the hydroxylamine to the stable nitroxide radical form in PEG-b-poly[4-(2,2,6,6-tetramethylpiperidine-1-oxyl)aminomethylstyrene] (PEG-b-PMNT), which shows an intense ESR signal. It is interesting to note that the ESR signal increased at a greater rate under acidic conditions (pH 5.6) than that under neutral conditions (pH 7.4), although the enzymatic activity of HRP under neutral conditions is known to be much higher than that under acidic conditions. This indicates that enzymatic oxidation reaction was accelerated by synchronizing the disintegration of HANP under acidic conditions. On the basis of these results, HANP can be used as a high-performance ESR probe for imaging of inflammation sites.

Application of optimized chemiluminescence assay for determination of the antioxidant capacity of herbal extracts

A chemiluminescence (CL) assay for the determination of antioxidant capacity (AOC) has been optimized and applied to analyses of herbal extracts in the present study. The optimal concentrations of reagents (luminol, H2O2, horseradish peroxidase) have been determined, as well as the optimal reaction conditions (wavelength, pH, temperature, sample volume). All of the measurements were performed at the emission maximum of the oxidized form of luminol (425 nm). The optimal concentrations of the reagents were determined as follows: 1.6 mmol/L luminol, 7.5 mmol/L H2O2 and 0.14 U/mL horseradish peroxidase activity in the reaction mixture. Analyses were carried out in phosphate buffer, pH 7.4, at room temperature. With the optimized CL assay, the AOCs of various water and methanol herbal extracts were determined (dog rose hips, plantain leaves and coltsfoot and thyme flowers) and the results were compared to those obtained by other classical methods for the evaluation of antioxidants. Strong correlations (r > 0.9) with the Folin-Ciocalteau assay and the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH)(●) assay are confirmed, although there is no correlation between AOC and the concentration of ascorbic acid in the samples analysed. This optimized CL assay is simple, rapid and reliable, and it represents a good alternative to classical methods (Folin-Ciocalteau, DPPH(●)) for the determination of AOC of herbal extracts and other food samples.