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Abstract: Raspberry and blackberry cultivars were assayed for total phenol content by enzymatic method using immobilized horseradish peroxidase (HRP). The HRP was immobilized on the styrene–divinylbenzene–polygluteraldehyde (STY–DVB–PGA) beads. Total phenol content determined by immobilized and free enzymatic methods was compared with those obtained by applying the Folin–Ciocalteu method. The total phenol content by immobilized HRP method ranged from 254 to 973mg gallic acid equivalent (GAE) per 100g fw as well as 105 to 435mg ellagic acid equivalent (EAE) per 100g fw in raspberry and blackberry cultivars. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical values were correlated with total phenol content in raspberry using immobilized enzymatic method as GAE (R 2 =0.7450, P =0.05) and EAE (R 2 =0.7449, P =0.05). All antioxidant activity values were highly correlated with total phenol content in blackberry using free and immobilized enzymatic methods as GAE (R 2 =0.9777 and 0.9223, P =0.05 respectively) and EAE (R 2 =0.9979 and 0.9223, P =0.05 respectively). A significant additivity was observed using individual phenolic compounds by immobilized enzymatic method as 95% (GAE) and 90% (EAE). These results show that the immobilized enzymatic assay can be applied to determine the total phenol content of berry fruits, which is more specific and not affected by interfering compounds. [Copyright &y& Elsevier]

Abstract: The interactions of heme peroxidase enzymes with their substrates have been studied for many years, but only in the last decade or so has structural information begun to appear. This review looks at crystal structures for a number of heme peroxidases in complex with a number of (mainly organic) substrates. It examines the nature and location of the binding interaction, and explores functional similarities and differences across the family. [Copyright &y& Elsevier]

Abstract: The shark antigen-binding VNAR domain has the potential to provide an attractive alternative to traditional biotherapeutics based on its small size, advantageous physiochemical properties, and unusual ability to target clefts in enzymes or cell surface molecules. The VNAR shares many of the properties of the well-characterised single-domain camelid VHH but is much less understood at the molecular level. We chose the hen-egg-lysozyme-specific archetypal Type I VNAR 5A7 and used ribosome display in combination with error-prone mutagenesis to interrogate the entire sequence space. We found a high level of mutational plasticity across the VNAR domain, particularly within the framework 2 and hypervariable region 2 regions. A number of residues important for affinity were identified, and a triple mutant combining A1D, S61R, and G62R resulted in a K D of 460 pM for hen egg lysozyme, a 20-fold improvement over wild-type 5A7, and the highest K D yet reported for VNAR–antigen interactions. These findings were rationalised using structural modelling and indicate the importance of residues outside the classical complementarity determining regions in making novel antigen contacts that modulate affinity. We also located two solvent-exposed residues (G15 and G42), distant from the VNAR paratope, which retain function upon mutation to cysteine and have the potential to be exploited as sites for targeted covalent modification. Our findings with 5A7 were extended to all known NAR structures using an in-depth bioinformatic analysis of sequence data available in the literature and a newly generated VNAR database. This study allowed us to identify, for the first time, both VNAR-specific and VNAR/Ig VL/TCR Vα overlapping hallmark residues, which are critical for the structural and functional integrity of the single domain. Intriguingly, each of our designated VNAR-specific hallmarks align precisely with previously defined mutational ‘cold spots’ in natural nurse shark cDNA sequences. These findings will aid future VNAR engineering and optimisation studies towards the development of VNAR single-domain proteins as viable biotherapeutics. [Copyright &y& Elsevier]

Abstract: The factors responsible for up-regulation of PTP1B, a negative regulator of insulin signaling, in insulin resistance state are not well understood. We performed a series of experiments in C2C12 muscle cells to determine the role of palmitate and an inflammatory state in regulation of PTP1B. Palmitate (0.75mM) induced PTP1B mRNA and protein level only at 16h. The combination of palmitate and macrophages, accompanied by a great increase of TNF-α and IL-6 in the culture media, additively caused a higher level of PTP1B protein levels in the muscle. Higher concentrations of palmitate reduced insulin stimulated glucose uptake in myotubes. A specific inhibitor of PTP1B partly increased insulin stimulated glucose uptake in palmitate treated cells. In conclusion, our results showing the additive influence of palmitate and the inflammatory state in the expression of PTP1B imply the involvement of these factors in the overexpression of PTP1B in insulin resistance state. We further provided the evidence suggesting the mediatory role for PTP1B in palmitate induced insulin resistance in myotubes. [Copyright &y& Elsevier]

Abstract: This article describes a simple and inexpensive signal amplification method, termed polymeric enzyme detection (PED), which permits rapid and sensitive detection of conserved sequences in the tuf gene that identify Staphylococcus genus, conserved sequences in the femB gene that specifically detect Staphylococcus aureus species, and the methicillin resistance gene mecA directly from positive blood culture bottles. Microbe-specific capture probes were immobilized onto microtiter plates or silicon chips. Target sequences and biotin-labeled, target-specific probes were hybridized to complementary capture probes to create a biotin-labeled, surface-immobilized tripartite complex. In a two-step process, signal was amplified by incubating the surface-immobilized biotin with streptavidin followed by the addition of a 500-kDa dextran polymer conjugated with approximately 80 biotins. Signal was then developed by binding of a streptavidin–horseradish peroxidase conjugate followed by incubation with the substrate tetramethylbenzidine. Use of the PED method improved the lower limit of detection 10- to 100-fold in model DNA hybridization assays with limits of detection as low as 1fmol/L target DNA. This level of sensitivity permits detection of genomic DNA from methicillin-resistant S. aureus positive blood cultures within 25 to 35min using either a thin film biosensor chip or a microtiter plate-based assay. [Copyright &y& Elsevier]

Abstract: Macromolecules that can assemble a large number of enzyme and antibody molecules have been used frequently for improvement of sensitivities in enzyme-linked immunosorbent assays (ELISAs). We generated bionanocapsules (BNCs) of approximately 30nm displaying immunoglobulin G (IgG) Fc-binding ZZ domains derived from Staphylococcus aureus protein A (designated as ZZ-BNC). In the conventional ELISA using primary antibody and horseradish peroxidase-labeled secondary antibody for detecting antigen on the solid phase, ZZ-BNCs in the aqueous phase gave an approximately 10-fold higher signal. In Western blot analysis, the mixture of ZZ-BNCs with secondary antibody gave an approximately 50-fold higher signal than that without ZZ-BNCs. These results suggest that a large number of secondary antibody molecules are immobilized on the surface of ZZ-BNCs and attached to antigen, leading to the significant enhancement of sensitivity. In combination with the avidin–biotin complex system, biotinylated ZZ-BNCs showed more significant signal enhancement in ELISA and Western blot analysis. Thus, ZZ-BNC is expected to increase the performance of various conventional immunoassays. [Copyright &y& Elsevier]

Abstract: A new enzymatic electrochemical biosensor based on disposable transducers, namely screen-printed carbon electrodes, has been developed for the determination of the antiepileptic drug levetiracetam. Horseradish peroxidase was immobilized onto the carbon working electrode previously modified by an aryl diazonium salt. The formation of amide bonds between the amino and carboxylic groups of the enzyme surface, catalyzed by hydroxysuccinimide and carbodiimide, leads to the electrode functionalization. This orientated enzymatic modification results in high reproducibility, with an associated relative standard deviation of 6.21% for the slopes of several calibration curves in the calibration range from 0.10 to 0.83mM. Experimental variables that can affect levetiracetam chronoamperometric response, such as hydrogen peroxide concentration, pH, and applied potential, were optimized to perform a selective determination. An average limit of detection of 1.75×10−5 M (α = β =0.05) was obtained. The biosensors were finally applied to the determination of levetiracetam in complex matrices such as pharmaceutical drugs, yielding successful results. [Copyright &y& Elsevier]

Abstract: The physical immobilization behavior of horseradish peroxidase (HRP) on cellulosic fiber surfaces was characterized using adsorption and inactivation isotherms measured by the depletion method followed by fitting of Langmuir’s and Freundlich’s models to the experimental data. The adsorption and inactivation behavior of simpler and relatively non-porous high and low crystalline cellulosic substrates (microcrystalline cellulose and regenerated cellulose) as well as more complex and porous cellulosic pulp fibers (bleached kraft softwood fibers) were investigated. The effect of the sorbent surface energy on HRP adsorption was demonstrated by increasing the hydrophobicity of the cellulosic fibers using an internal sizing agent. The influence of the fiber surface charge density on HRP adsorption was studied via modification of the cellulosic fibers using TEMPO (2,2,6,6-tetramethyl-1-piperidiniloxy radical)-mediated oxidation methods. Results showed that hydrophobic interactions had a much larger effect on HRP adsorption than electrostatic interactions. More hydrophobic fiber surfaces (lower polar surface energy) result in larger enzyme-fiber binding affinity constants and higher binding heterogeneity. It was also found that oxidation of the cellulosic fiber substrate reduces enzyme adsorption affinity but significantly increases the loading capacity per unit weight of the surface. [Copyright &y& Elsevier]

Abstract: An anti-oxidative plastic film coated with microcapsules containing volatile horseradish extract was developed. The oil-in-water-type microcapsule was produced by a modified orifice method to encapsulate the methanol extract of horseradish. The presence of naturally occurring antioxidants in the extract was confirmed by high performance liquid chromatography. Increasing the amount of chitosan during microcapsule formation increased the size of the microcapsules and decreased the rate of release of the horseradish extract, suggesting that the amount of extract released from the film can be modulated by the chitosan content of the microspheres. Covering pork and fish fillets with the anti-oxidative film delayed oxidative discoloration and rancidization. Collectively, these results showed that a horseradish-coated film containing natural antioxidants efficiently enhanced the stability of both pork and fish. This novel film may be a promising tool to prolong the shelf-life of meats. [Copyright &y& Elsevier]

Abstract: Polypyrrole was synthesized in high yield by a biocatalytic method in mild aqueous media using hydrogen peroxide as oxidizer. A redox mediator, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) diammonium salt, was used to oxidize the pyrrole. ABTS is a very effective peroxidase substrate, which was enzymatically oxidized to generate a radical cation that in turn was able to chemically oxidize pyrrole. This indirect biocatalytic method was implemented because pyrrole is not a substrate of horseradish peroxidase, however, the polymerization process was successfully optimized and later adapted to prepare also polypyrrole thin films and water dispersible polypyrrole colloids. The polypyrrole powder and colloids were characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, electrical conductivity, and thermogravimetric analysis. In addition, the deposition of the polypyrrole thin film was monitored using a quartz-crystal microbalance and its morphology studied by optical and scanning electron microscopy. The biocatalytic polymerization of pyrrole results in a polymer spectroscopically very similar to chemically synthesized polypyrrole. [Copyright &y& Elsevier]

Abstract: This study aims to establish model systems for the exploration of proximity effects, occurring in natural multienzyme complexes. DNA-directed assembly of covalent conjugates of DNA oligonucleotides and Glucose Oxidase (GOX) or Horseradish peroxidase (HRP) was used to generate supramolecular complexes, in which the two enzymes were arranged with defined spatial orientation. Electrophoretic studies indicated that the assembly efficiency significantly depends on positional and sterical factors of the two DNA–enzyme conjugates. Kinetic rate measurements of the coupled reaction of glucose oxidation and Amplex Red peroxidation were carried out with microplate-immobilized DNA–GOX–HRP complexes, and the influence of Catalase on this reaction was determined. The kinetic measurements revealed a significant increase in the reactivity of the complexes, in which GOX and HRP were immobilized in direct proximity on a complementary DNA carrier. [Copyright &y& Elsevier]

Abstract: Accumulating evidence indicates that post-translational protein modifications by nitric oxide and its derived species are critical effectors of redox signaling in cells. These protein modifications are most likely controlled by intracellular reductants. Among them, the importance of the 12 kDa dithiol protein thioredoxin-1 (TRX-1) has been increasingly recognized. However, the effects of TRX-1 in cells exposed to exogenous nitrosothiols remain little understood. We investigated the levels of intracellular nitrosothiols and survival signaling in HeLa cells over-expressing TRX-1 and exposed to S-nitrosoglutahione (GSNO). A role for TRX-1 expression on GSNO catabolism and cell viability was demonstrated by the concentration-dependent effects of GSNO on decreasing TRX-1 expression, activation of caspase-3, and increasing cell death. The over-expression of TRX-1 in HeLa cells partially attenuated caspase-3 activation and enhanced cell viability upon GSNO treatment. This was correlated with reduction of intracellular levels of nitrosothiols and increasing levels of nitrite and nitrotyrosine. The involvement of ERK, p38 and JNK pathways were investigated in parental cells treated with GSNO. Activation of ERK1/2 MAP kinases was shown to be critical for survival signaling. In cells over-expressing TRX-1, basal phosphorylation levels of ERK1/2 MAP kinases were higher and further increased after GSNO treatment. These results indicate that the enhanced cell viability promoted by TRX-1 correlates with its capacity to regulate the levels of intracellular nitrosothiols and to up-regulate the survival signaling pathway mediated by the ERK1/2 MAP kinases. [Copyright &y& Elsevier]

Abstract: Interleukin (IL) 22 is a type II cytokine that is produced by immune cells and acts on nonimmune cells to regulate local tissue inflammation. As a product of the recently identified T helper 17 lineage of CD4 + effector lymphocytes, IL-22 plays a critical role in mucosal immunity as well as in dysregulated inflammation observed in autoimmune diseases. We used comprehensive mutagenesis combined with mammalian cell expression, ELISA cell-based, and structural methods to evaluate how IL-22 interacts with its cell surface receptor, IL-22R/IL-10R2, and with secreted IL-22 binding protein. This study identifies those amino acid side chains of IL-22 that are individually important for optimal binding to IL-22R, considerably expands the definition of IL-22 surface required for binding to IL-10R2, and demonstrates how IL-22 binding protein prevents IL-22R from binding to IL-22. The IL-22R and IL-10R2 binding sites are juxtaposed on adjacent IL-22 surfaces contributed mostly by helices A, D, and F and loop AB. Our results also provide a model for how IL-19, IL-20, IL-24, and IL-26 which are other IL-10-like cytokines, interact with their respective cell surface receptors. [Copyright &y& Elsevier]

Abstract: The protein CsaA has been proposed to function as a protein secretion chaperone in bacteria that lack the Sec-dependent protein-targeting chaperone SecB. CsaA is a homodimer with two putative substrate-binding pockets, one in each monomer. To test the hypothesis that these cavities are indeed substrate-binding sites able to interact with other polypeptide chains, we selected a peptide that bound to CsaA from a random peptide library displayed on phage. Presented here is the structure of CsaA from Agrobacterium tumefaciens (AtCsaA) solved in the presence and absence of the selected peptide. To promote co-crystallization, the sequence for this peptide was genetically fused to the amino-terminus of AtCsaA. The resulting 1.65 Å resolution crystal structure reveals that the tethered peptide from one AtCsaA molecule binds to the proposed substrate-binding pocket of a symmetry-related molecule possibly mimicking the interaction between a pre-protein substrate and CsaA. The structure shows that the peptide lies in an extended conformation with alanine, proline and glutamine side chains pointing into the binding pocket. The peptide interacts with the atoms of the AtCsaA-binding pocket via seven direct hydrogen bonds. The side chain of a conserved pocket residue, Arg76, has an “up” conformation when the CsaA-binding site is empty and a “down” conformation when the CsaA-binding site is occupied, suggesting that this residue may function to stabilize the peptide in the binding cavity. The presented aggregation assays, phage-display analysis and structural analysis are consistent with AtCsaA being a general chaperone. The properties of the proposed CsaA-binding pocket/peptide interactions are compared to those from other structurally characterized molecular chaperones. [Copyright &y& Elsevier]

Abstract: In this study, a new chemiluminescence (CL) flow-through biosensor for glucose was developed by immobilizing glucose oxidase (GOD) and horseradish peroxidase (HRP) on the eggshell membrane with glutaraldehyde as a cross-linker. The CL detection involved enzymatic oxidation of glucose to d-gluconic acid and hydrogen peroxide (H2O2) and then H2O2 oxidizing luminol to produce CL emission in the presence of HRP. The immobilization condition (e.g., immobilization time, GOD/HRP ratio, glutaraldehyde concentration) was studied in detail. It showed good storage stability at 4°C over a 5-month period. The proposed biosensor exhibited short response time, high sensitivity, easy operation, and simple sensor assembly, and the proposed biosensor was successfully applied to the determination of glucose in human serum. [Copyright &y& Elsevier]

Abstract: A disposable pseudo-mediatorless amperometric biosensor has been fabricated for the determination of hydrogen peroxide (H2O2). In the current study, an indium–tin oxide (ITO) electrode was modified with thiol functional group by (3-mercaptopropyl)trimethoxysilane. The stable nano-Au-SH monolayer (AuS) was then prepared through covalent linking of gold nanoparticles and thiol groups on the surface of the ITO. The horseradish peroxidase (HRP) and tetramethyl benzidine (TMB) were finally coentrapped by the colloidal gold nanoparticles. The immobilized TMB was used as an electron transfer mediator that displayed a surface-controlled electrode process at a scan rate of less than 50mV/s. The biosensor was characterized by photometric and electrochemical measurements. The results showed that the prepared AuS monolayer not only could steadily immobilize HRP but also could efficiently retain HRP bioactivity. Parameters affecting the performance of the biosensor, including the concentrations of the immobilized TMB and HRP, the pH value, and the reaction temperature, were optimized. Under the optimized experimental conditions, H2O2 could be determined in a linear calibration range from 0.005 to 1.5mM with a correlation coefficient of 0.998 (n =14) and a detection limit of 1μM at a signal/noise ratio of 3. The proposed method provides a new alternative to develop low-cost biosensors by using ITO film electrodes from industrial mass production. [Copyright &y& Elsevier]

Abstract: As an alternative to radioimmunoassays, a simple, highly sensitive and quick enzymeimmunoassay (EIA) for determination of cortisol in blood plasma of yaks on microtiterplates using second antibody coating technique and cortisol–horseradish peroxidase as a label has been developed. The wells of the microtiterplate were coated with affinity-purified goat IgG (antirabbit IgG) that binds the hormone specific antibody. The EIA was carried out directly in 20μl of heat treated plasma after 1:5 dilution with PBS. The cortisol standard curve, with doses ranged from 0.4 to 100pg/well. The sensitivity of the assay was 20pg/ml. Cortisol standard curve in buffer showed parallelism with serially diluted yak plasma containing high endogenous cortisol. Intra- and inter-assay coefficients of variation (CV) determined using pooled plasma was found 6.58 and 11.35%, respectively. Recovery of known concentrations of added cortisol in charcoal stripped plasma was linear (r =0.98). For biological validation of cortisol enzymeimmunoassay, the blood samples were collected from yak cows at −48 and −24h before and 0, 12, 24, 36, 48, 60, 72, 84 and 96h after dexamethasone administration. The plasma cortisol before dexamethasone administration was significantly (P <0.05) higher than after dexamethasone administration. The developed EIA was further validated biologically by estimating cortisol in peri-parturient cows beginning day 10 prior to calving till day 10 post-calving; the concentrations were along with the expected lines as reported in bovine. In conclusion, the EIA developed in this study is simple, highly sensitive, valid and sufficiently reliable method for estimation of cortisol directly in bovine plasma. [Copyright &y& Elsevier]

Abstract: Influence of unsaturation present at the surfactant head on the activity of interfacially located enzyme, lipase, and horseradish peroxidase (HRP) is investigated in cationic reverse micelles of a series of surfactants having unsaturated (allyl and pyridinium moieties) as well as analogous saturated (n-propyl and piperidinium moieties) polar head. Lipase activity increases with n-propyl (saturated) substitution as the increase in the headgroup area (A min) presumably provides greater space for the enzyme to attain flexible conformation and increases the local concentrations of enzyme and substrate at the interface. In contrast, activity of lipase decreases with increasing number of allyl (unsaturated) substitution though A min gradually increased. Similar trend in deactivation was observed when unsaturation is present in cyclic ring (pyridine) at the surfactant head in comparison to the saturated analogue, piperidine. Circular dichroism (CD) spectra of lipase in reverse micelles indicate that ellipticity in the far-UV region increases with increasing unsaturation. Thus, lipase probably loses its α-helix content and thereby its activity. Inhibition of biocatalyst with increasing unsaturation at the polar head of surfactant is also observed in case of HRP, an oxidoreductase enzyme. [Copyright &y& Elsevier]

Abstract: The hydrogen donor couples pyrocatechol–aniline and phenol–aminoantipyrine in the presence of hydrogen peroxide were compared as chromogens for horseradish peroxidase (HRP) assay. UV–Visible spectroscopy and high-performance liquid chromatography analysis indicated that during the HRP biocatalytic process, pyrocatechol–aniline was converted to a pink-colored reagent with a λ max of 510nm, which was used in the assay of HRP activity. Electrochemical studies revealed adequate electron transfer ability for this color reagent to serve as a proper mediator for HRP also. Using pyrocatechol–aniline a higher sensitivity and lower detection limit was obtained relative to those of the phenol–aminoantipyrine couple, which is commonly used for HRP assay. A relative standard deviation of 2.9% was obtained for 20 HRP activity measurements, indicating a satisfactory reproducibility for this method. In addition, kinetic parameters of K m (12.5mM) and V max (12.2mMmin−1 mg−1) were calculated for pyrocatechol–aniline. Regarding the superiority of pyrocatechol–aniline, this couple is suggested to be a better hydrogen donor for the HRP spectrophotometric assay. [Copyright &y& Elsevier]

Abstract: A number of peptide tags are available to facilitate the characterization of recombinant proteins. We have tested the bacterial oxaloacetate decarboxylase biotinylation domain for its efficacy in tagging recombinant proteins in vivo in Leishmania. To achieve efficient biotinylation, Leishmania also had to be co-transformed with the gene for bacterial biotin protein ligase (birA gene product). The recombinant chimeric protein could be detected on blots probed with avidin–horseradish peroxidase and purified on immobilized monomeric avidin resins. [Copyright &y& Elsevier]

Abstract: Laboratory diagnostics of toxoplasmosis depends primarily on serological methods detecting specific antibodies. Since these methods do not always enable specific and sensitive recognition of the infection and phase of toxoplasmosis, the search for new diagnostic tools continues. Recombinant antigens promise a new alternative in diagnostics of Toxoplasma gondii infections. In this work the usefulness of six recombinant T. gondii antigens: GRA1, GRA6, GRA7, p35, SAG1, and SAG2 in the detection of primary murine toxoplasmosis was evaluated. Sera obtained from infected mice differing in their natural susceptibility to T. gondii infection, BALB/c (relatively resistant) and C57BL/6 (relatively susceptible), were tested using ELISA. During acute infection high response to GRA7, GRA6, and p35 antigens was noticed, whereas a strong reactivity with surface antigens SAG1 and SAG2 was characteristic for chronic toxoplasmosis. Our results show that the recombinant antigens are useful in distinguishing between acute and chronic toxoplasmosis regardless of the genetically determined susceptibility of the host. [Copyright &y& Elsevier]

Abstract: Plant peroxidases, as typified by horseradish peroxidase (HRP), primarily catalyze the one-electron oxidation of phenols and other low oxidation potential substrates. In contrast, the mammalian homologues such as lactoperoxidase (LPO) and myeloperoxidase primarily oxidize halides and pseudohalides to the corresponding hypohalides (e.g., Br− to HOBr, Cl− to HOCl). A further feature that distinguishes the mammalian from the plant and fungal enzymes is the presence of two or more covalent bonds between the heme and the protein only in the mammalian enzymes. The functional roles of these covalent links in mammalian peroxidases remain uncertain. We have previously reported that HRP can oxidize chloride and bromide ions, but during oxidation of these ions undergoes autocatalytic modification of its heme vinyl groups that virtually inactivates the enzyme. We report here that autocatalytic heme modification during halide oxidation is not unique to HRP but is a general feature of the oxidation of halide ions by fungal and plant peroxidases, as illustrated by studies with Arthromyces ramosus and soybean peroxidases. In contrast, LPO, a prototypical mammalian peroxidase, is protected from heme modification and its heme remains intact during the oxidation of halide ions. These results support the hypothesis that the covalent heme-protein links in the mammalian peroxidases protect the heme from modification during the oxidation of halide ions. [Copyright &y& Elsevier]

The highly sensitive, convenient fluorescence assay, based on the oxidation of nonfluorescent 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) to highly fluorescent resorufin, is becoming increasingly popular for hydrogen peroxide quantitation. Yet, the intricacies of the horseradish peroxidase-catalyzed oxidation of the reductant substrate Amplex Red by hydrogen peroxide and the resulting resorufin could complicate the assay design and data interpretation. In particular, substrate inhibition and enzyme inactivation at higher hydrogen peroxide concentrations were known to affect the enzyme kinetics and end-point fluorescence. In addition, here we report the spontaneous transformation of resorufin to less or nonfluorescent product(s) in the absence of hydrogen peroxide and horseradish peroxidase. This spontaneous decay of resorufin fluorescence is most prominent in the pH range 6.2–7.7, likely due to general base-catalyzed de-N-acetylation and polymerization of resorufin. From a practical point of view, precautions for properly designing assays for hydrogen peroxide or characterizing hydrogen peroxide-generating systems are discussed based on the spontaneous transformation of resorufin to less fluorescent compound(s), substrate inhibition and enzyme inactivation at higher (>100μM) hydrogen peroxide concentrations, and enzymatic oxidation of resorufin to nonfluorescent resazurin. [Copyright &y& Elsevier]

We report here that reduced pyridine nucleotides and reduced glutathione result in an oxidation of Amplex Red by dioxygen that is dependent on the presence of horseradish peroxidase (HRP). Concentrations of NADH and glutathione typically found in biological systems result in the oxidation of Amplex Red at a rate comparable to that produced, for example, by respiring mitochondria. The effects of NADH and glutathione in this assay system are likely to be the result of H2O2 generation via a superoxide intermediate because both catalase and superoxide dismutase prevent the oxidation of Amplex Red. These results suggest caution in the assay of H2O2 production in biological systems using the Amplex Red/HRP because the assay will also report the mobilization of NADH or glutathione. However, the interruption of this process by the addition of superoxide dismutase offers a simple and reliable method for establishing the source of the oxidant signal. [Copyright &y& Elsevier]

Wasabi (Japanese horseradish) is used extensively in Japanese cuisine because of its characteristic flavour. The plant is grown as a perennial crop in New Zealand. The rhizomes are the most sought after part of wasabi although other parts of the plants are pungent. In this study the yields of individual and total isothiocyanates (ITCs) were measured in the four main parts of the plant: root, rhizome, petiole and leaves in mature, flowering and non-flowering, 18-month-old plants. The plants were grown without fertilizer in beds flooded with river water. The total ITC and individual contents of allyl, 3-butenyl, 4-pentenyl and 5-hexenyl ITC in the roots and rhizomes of the flowering plants were all significantly higher (P<0.001) than in the comparable non-flowering plant parts. The level of total ITC in the epidermis and cortex of rhizomes of the flowering plants was 3144 mg/kg fresh weight basis compared to 1773 mg/kg in the non-flowering tissue. The total ITC contained in the vascular and pith tissue was 2234 mg/kg fresh weight for the flowering plants compared to 1388 mg/kg fresh weight for the non-flowering plants. Such differences between the ITC profiles in the flowering and non-flowering tissue of wasabi plants suggest there will be differences in the overall aroma profiles when the material is made into a processed product. The magnitude of such differences suggest the flowering state of wasabi plants could be important in crop management for optimal processing quality. [Copyright &y& Elsevier]

Abstract: We describe a procedure for detection and comparison of protein–DNA interactions using DNA–BIND plate and horseradish peroxidase (HRP)-based colorimetric assay. Amino-modified oligonucleotide was covalently immobilized on the surface of DNA–BIND plate. After the complementary oligonucleotide was annealed, the plate was incubated with protein to allow sequence-specific DNA binding. Primary antibody and HRP-labeled secondary antibody were then employed, and colorimetric assay was conducted before the absorbance was read. This is a sensitive, specific, and high-throughput method that has been applied not only in the detection of protein–DNA interaction but also in the quantitative comparison of DNA-binding capabilities among wild-type and mutant proteins. [Copyright &y& Elsevier]