• Molecules and Cells
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• v.21 no.1
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• pp.161-165
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• 2006

Dichlorodihydrofluorescein ($DCFH_2$) is a widely used probe for intracellular $H_2O_2$. However, $H_2O_2$ can oxidize $DCFH_2$ only in the presence of a catalyst, whose identity in cells has not been clearly defined. We compared the peroxidase activity of Cu,Zn-superoxide dismutase (CuZnSOD), cytochrome c, horseradish peroxidase (HRP), $Cu^{2+}$, and $Fe^{3+}$ under various conditions to identify an intracellular catalyst. Enormous increase by bicarbonate in the rate of $DCFH_2$ oxidation distinguished CuZnSOD from cytochrome c and HRP. Cyanide inhibited the reaction catalyzed by CuZnSOD but accelerated that by $Cu^{2+}$ and $Fe^{3+}$. Oxidation of $DCFH_2$ by $H_2O_2$ in the presence of a cell lysate was also enhanced by bicarbonate and inhibited by cyanide. Confocal microscopy of $H_2O_2$-treated cells showed enhanced DCF fluorescence in the presence of bicarbonate and attenuated fluorescence for the cells pre-incubated with KCN. Moreover, DCF fluorescence was intensified in CuZnSOD-transfected HaCaT and RAW 264.7 cells. We propose that CuZnSOD is a potential intracellular catalyst for the $H_2O_2$-dependent oxidation of $DCFH_2$.

• KSBB Journal
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• v.14 no.1
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• pp.82-90
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• 1999

In order to eventually fabricate an analytical system for infectious microorganisms, we synthesized major immunochemical components, utilized them for the construction of model system, and investigated an assay concept for bacterial whole cells. For the preparation of system components, a polyclonal antibody, against Salmonella thompson as model analyte, purified by immuno-affinity chromatography was used to chemically link to streptavidin or an enzyme, horseradish peroxidase(HRP). The antibody and streptavidin was modified with sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate and N-succinimidyl-3-[2-pyridyldithio]propionate(subsequently activated by dithiotheritol), respectively. The modified components were reacted to synthesize antibody-streptavidin conjugates which were then purified on a two-layer chromatography column of diaminobiotin gel and Sephadex G-100. For antibody-HRP conjugates, HRP molecules were activated by $NalO_4$ oxidation and then coupled to immunoglobulin. After stabilizing with ($NaCNBH_3$, the conjugates were purified by size exclusion chromatography on Biogel A5M column. To devise a model system, such produced components were combined with a dot-blotter in which a nitrocellulose membrane($12{\mu}m$ pre size) with immobilized biotin was already located. The analyte (S. thompson cells) was reacted with the both antibody conjugates in a liquid phase, and the complexes formed were captured on the membrane surfaces by applying vacuum in the bottom compartment of the blotter to invoke biotin-streptavidin reaction. Under optimal conditions, the system enabled to identify the analytical concept for bacterial whole cells, and the lower limit of detection was approximately $1{\mu}g/m{\ell}$($10^5-10^6$ cells/m$m{\ell}$). The controlling factors were the concentrations of each antibody conjugate that caused agglutination in the presence of analyte as they increased.

• Archives of Pharmacal Research
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• v.21 no.3
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• pp.231-235
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• 1998

A sensitive enzyme immunoassay for serum TSH has been developed utilizing the tight binding between biotin and avidin, and three layered protein polystyrene beads as solid phase. To increase binding capacity of TSH and sensitivity of the assay, the polystyrene beads were coated sequentially with mouse immunoglobulin as first layer, rabbit antimouse immunoglobulin as second layer and monoclonal anti-TSH as third layer. A serum sample was incubated simultaneously with a monoclonal anti-TSH immobilized polystyrene beads and a second monoclonal anti-TSH covalently attached to biotin. After washing, the antibody bound serum TSH-anti-TSH-biotin complex is reacted with horseradish peroxidase (HRP)-labeled avidin. Following second wash, the bound HRP activity was measured calorimetrically. Reproducible results were obtained within 4 hours for serum TSH in the range between $0{\mu}\textrm{IU}$ml and ${50}{\mu}\textrm{IU}$ml with detection limit of $0.1{\mu}\textrm{IU}$ per test.

• Bulletin of the Korean Chemical Society
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• v.18 no.1
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• pp.44-50
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• 1997

A method of dual-signal generation from two different enzymes was developed and utilized to simultaneously perform dual immunoassays in a single microwell. Two enzymes selected as tracers were horseradish peroxidase (HRP) and β-galactosidase (GAL). 3, 3', 5, 5'-Tetramethylbenzidine (TMB) and chlorophenolred-β-galactopyranoside (CPRG) as chromogenic substrates for the respective enzyme were used. Although the two enzymes showed their maximum activities at distinct pH conditions (pH 5.1 for HRP and 7.5 for GAL), the enzyme reactions were able to be concurrently carried out at pH 5.75 in a dual-substrate solution without signal loss. This performance was achieved by increasing TMB concentration two-fold, introducing potassium salt as activator of GAL reaction, and extending total reaction time 50%. The signal generation method was then used for dual-enzyme immunoassays to detect antibodies with co-immobilized Hepatitis C virus antigens (core and NS5) and a Hepatitis B virus antigen (PreS(2)) in a microwell. Dose-response curves of the assays revealed cooperativity between different antigen-antibody complex formation, which suggested that dual immunoassays can only be used for qualitative screening tests unless the antigens immobilized were spatially separated.

• Nuclear Engineering and Technology
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• v.53 no.1
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• pp.142-147
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• 2021

Hydrogen peroxide is a radiolysis product of water formed under gamma-irradiation; therefore, its reliable detection is crucial in the nuclear industry for spent fuel management and coolant chemistry. This study proposes an electrochemical sensor for hydrogen peroxide detection. Cysteamine (CYST), gold nanoparticles (GNPs), and horseradish peroxidase (HRP) were used in the modification of a gold electrode for fabricating Au/CYST/GNP/HRP sensor. Each modification step of the electrode was investigated through electrochemical and physical methods. The sensor exhibited strong sensitivity and stability for the detection and measurement of hydrogen peroxide with a linear range of 1-9 mM. In addition, the Michaelis-Menten kinetic equation was applied to predict the reaction curve, and a quantitative method to define the dynamic range is suggested. The sensor is highly sensitive to H₂O₂ and can be applied as an electrochemical H₂O₂-sensor in the nuclear industry.

• The Korean Journal of Zoology
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• v.39 no.1
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• pp.26-35
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• 1996

The projection from the lateral reticular nucleus (LRN) to three subdivisions of the cerebellar vermal lobule VI was studied in the rat by utilizing the retrograde transport of wheatgerm agglutinin-conjugated horseradish peroxidase. Labelled neurons were located bilaterally throughout the LRN, but with ipsilateral predominance. There seemed to be a dorsal4o-ventral transition in the ipsilateral magnocellular neurons projecting to the cerebellar vermal lobules VIa-to-Vic. In the contralateral side, cells projecting to vermal lobule Via were observed at more rostral sections, whereas those projecting to lobule Vic were located at caudal sections. There were relatively few labelled neurons in parvocellular or subtrigeminal divisions at either side. Computer-aided three dimensional reconstruction of LRN projections to lobules Via/Vib or lobules Vib/Vic exhibited extensive oberlap within each combination of injection cases.

• Journal of Microbiology and Biotechnology
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• v.13 no.5
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• pp.773-777
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• 2003

The color of Congo red hinders the spectrometric measurements of a concentration of hydrogen peroxide and enzyme activity (Horseradish peroxidase; HRP) during enzymatic decoloring of Congo red. In this study, a method was developed to measure peroxidase activity and hydrogen peroxide concentration in the presence of Congo red. The oxidation product of HRP/hydrogen peroxide and ABTS(2,2'-azino-bis-(3-ethylbenzotriazoline-6-sulfonic acid)) formed a dark green color. The spectrum of this product showed absorption bands at 420 nm and 734 nm. When compared with the Congo red spectrum, the absorption at 734 nm of this product did not overlap with Congo red, thus making the hydrogen peroxide measurement possible even in the presence of Congo red. Kinetic study of decoloring of Congo red performed by this method showed that the decoloring reaction followed the Michaelis-Menten kinetics. Pulse feeding of hydrogen peroxide, upon depletion, significantly increased the decoloring of Congo red. This result shows that this newly developed technique can monitor, predict, and improve the enzymatic decoloring process.

• Journal of the Korean Wood Science and Technology
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• v.38 no.3
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• pp.223-229
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• 2010

In this study diverse dehydrogenative polymers (DHPs) were synthesized with three precursors of native lignin [p-coumaryl alcohol (PCA), coniferyl alcohol (CA), sinapyl alcohol (SA)] in the presence of horseradish peroxidase (HRP, EC. 1.11.1.7)/$H_2O_2$. To compare the structural features between DHPs and native lignin, the DHPs as well as pine/poplar milled wood lignins were simultaneously subjected to gel permeation chromatography (GPC) to determine average molecular weights and derivatization followed by reductive cleavage (DFRC) to investigate the frequency of ${\beta}$-O-4 linkage. The highest yield of DHP was measured to 71% when CA was solely injected (G-DHP) and the yield of H-DHP was 42%. However, single injection of SA could not form any polymer in this system. The average molecular weights of DHPs were ranged between 3,000~4,700, which were only 1/2 fold compared with that of pine MWL (G-type lignin: Mw 7,340) and 1/3 scale compared with that of poplar MWL (GS-type lignin: Mw 13,250). DFRC analysis revealed that the formation of ${\beta}$-O-4 linkage during dehydrogenative polymerization was the highest in the GS-DHP with ca. 502 ${\mu}mol$/g, which was, however, remained to only 50% compared to that in poplar MWL (1107 ${\mu}mol$/g). The ${\beta}$-O-4 linkage was estimated to ca. 286 ${\mu}mol$/g In the G-DHP, which was twice as much as that of H-DHP(127 ${\mu}mol$/g). Similar to GS-DHP, only half amount of ${\beta}$-O-4 linkage, compared to pine MWL, was formed during in vitro polymerization of CA by horseradish peroxidase/$H_2O_2$.

• Journal of Microbiology and Biotechnology
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• v.14 no.4
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• pp.686-692
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• 2004

To develop the enzyme-linked immunosorbent assay (ELISA) for the analysis of N-acetylehitooligosaccharides (NACOS) and chitooligosaccharides (COS), specific antibodies (Abs) were produced, and their properties were compared. N-acetylehitohexaose (NACOS6), chitohexaose (COS6), and COS mixture (COSM) conjugated to bovine serum albumin (BSA) were used to immunize rabbits. By the use of specific Abs and NACOS6-horseradish peroxidase (HRP), COS6-HRP, and COSM-HRP conjugates, competitive direct ELISA (cdELISA) was developed. The detection limits of NACOS6 by the anti-NACOS6 Ab and COS6 by the anti-COS6 and the anti-COSM Abs in the cdELISAs were about 0.2, 2, ana 2 ng/ml (ppb), respectively. In the cdELISA, the anti-NACOS6 Ab was found to recognize NACOS3-NACOS6, but not N-acetyl-D-glucosamine (GlcNAc), NACOS2, and COSs; the anti-COS6 Ab recognized COS2-COS6 and COSM, but not glucosamine (GlcN) and NACOSs. The recognition pattern of the anti-COSM Ab was almost the same as that of the anti- COS6 Ab, except that the former recognized COS2 and COS3 slightly better than the latter.

• YAKHAK HOEJI
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• v.35 no.6
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• pp.486-496
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• 1991

Spinal parasympathetic outflows originate in the sacral parasympathetic nuclei. The sacral parasympathetic nuclei receive inputs from the brainstem. Many areas in the medulla appear to influence sympathetic outflow of the spinal cord. Whether neurons in these areas of the medulla may project to the lumbosacral cord to affect the parasympathetic outflow has not been studied clearly. Thus, this study was intended to investigate origins of cells projecting from the medulla to the sacral parasympathetic nuclei of the spinal cord. In 3 cats, horseradish peroxidase (HRP) was injected into the lower lumbar spinal cord. HRP labeled neurons were found mainly in the following areas: nucleus retroambiguus, nucleus tractus solitarius, raphe complex and ventrolateral area of the rostral medulla. Most of these areas are known to be involved in regulation of sympathetic activity, and, thus, these results indicate that these areas are likely to affect the sacral parasympathetic outflow as they do for the sympathetic nerves.