• Title, Summary, Keyword: Stopped flow

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Enhancing Effect of Organic Substances on Hydroxyl Radical Generation During Ozonation of Water: Stopped-Flow ESR Technique

  • Han, Sang-Kuk
    • Bulletin of the Korean Chemical Society
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    • v.25 no.12
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    • pp.1907-1910
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    • 2004
  • Generation of hydroxyl radical, one of their major active species in ozonation of water was directly observed with spin-trapping/electron spin resonance (ESR) technique using 5,5-dimethyl-pyrrolidine-1-oxyl (DMPO) as a spin-trapping reagent. Hydroxyl radical was trapped with DMPO as a stable radical, DMPO-OH. 80 mM of ozone produced $1.08{\times}10^{-6}$M of DMPO-OH, indicating that 1.4% of ${\cdot}$OH is trapped with DMPO if ${\cdot}$OH is produced stoichiometrically from ozone. Humic acid suppressed DMPO-OH generation in a dose-dependent manner. Generation rate of DMPO-OH was determined with ESR/stopped-flow measurement. Phenol derivatives increased the amount and generation rate of DMPO-OH, indicating that phenol derivatives enhance·OH generation during ozonation of water.

Rapid Measurement of $NH_3$ and Weak Acid Permeation Through Liposomes and Renal Proximal Tubule Membranes

  • Bae, Hae-Rahn;Suh, Duck-Joon;Lee, Sang-Ho
    • The Korean journal of physiology & pharmacology
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    • v.28 no.2
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    • pp.203-214
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    • 1994
  • Using the methods of stopped-flow and epifluorescence microscopy with entrapped fluorophore, membrane permeability of $NH_3$ and weak acids in liposomes, renal brush border (BBMV) and basolateral membrane vesicles (BLMV), and primary culture cells from renal proximal tubule was measured. Permeability coefficient (cm/sec) of $NH_3$ was $(2.9{\times}10^{-2}$ in phosphatidylcholine liposome $25^{\circ}C)$, $5.9{\times}10^{-2}$ in renal proximal tubule cell $(37^{\circ}C)$, $4.0{\times}10^{-2}\;and\;2.4{\times}10^{-2}$ in BBMV and BLMV $(25^{\circ}C)$, respectively. Formic acid has the highest permeability coefficient among the weak acids tested, which was $4.9{\times}10^{-3}$ in liposome, $5.0{\times}10^{-3}$ in renal proximal tubule cell, $9.1{\times}10^{-3}$ in BBMV and $3.8{\times}10^{-3}$ in BLMV. There was a linear relationship between external concentration of nonionized formic acid and initial rate of flux of formic acid in liposome, and the slope coincided with the value of permeability coefficient of formic acid measured in pH 7.0. These results show that techniques of stopped-flow and epifluorescence microscopy with entrapped fluorophore provide the precise method of measurement of very rapid transport of nonelectrolytes through membranes with the advantages of instantaneous mixing effect, good resolution time and easy manipulation.

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Kinetic Mechanism of Nucleotide Binding to Escherichia coli Transcription Termination Factor Rho: Stopped-flow Kinetic Studies Using ATP and Fluorescent ATP Analogues

  • Kim, Dong-Eun
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.9 no.1
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    • pp.23-34
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    • 2004
  • Escherichia coli transcription termination factor Rho catalyzes the unwinding of RNA/DNA duplex in reactions that are coupled to ATP binding and hydrolysis. Fluorescence stopped-flow methods using ATP and the fluorescent 2'(3')-O-( N-methylanthraniloyl) derivatives (mant-derivatives) of ATP and ADP were used to probe the kinetics of nucleotide binding to and dissociation from the Rho-RNA complex. Presteady state nucleotide binding kinetics provides evidence for the presence of negative cooperativity in nucleotide binding among the multiple nucleotide binding sites on Rho hexamer. The binding of the first nucleotide to the Rho-RNA complex occurs at a bimolecular rate of 3.6${\times}$10$\^$6/ M$\^$-1/ sec$\^$-1/ whereas the second nucleotide binds at a slower rate of 4.7${\times}$10$\^$5/ M$\^$-1/ sec$\^$-1/ at 18$^{\circ}C$, RNA complexed with Rho affects the kinetics of nucleotide interaction with the active sites through conformational changes to the Rho hexamer, allowing the incoming nucleotide to be more accessible to the sites. Adenine nucleotide binding and dissociation is more favorable when RNA is bound to Rho, whereas ATP binding and dissociation step in the absence of RNA occurs significantly slower, at a rate ∼70- and ∼40-fold slower than those observed with the Rho-RNA complex, respectively.

Critical Role of Glu175 on Stability and Folding of Bacterial Luciferase: Stopped-flow Fluorescence Study

  • Shirazy, Najmeh Hadizadeh;Ranjbar, Bijan;Hosseinkhani, Saman;Khalifeh, Khosrow;Madvar, Ali Riahi;Naderi-Manesh, Hossein
    • BMB Reports
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    • v.40 no.4
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    • pp.453-458
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    • 2007
  • Bacterial luciferase is a heterodimeric enzyme, which catalyzes the light emission reaction, utilizing reduced FMN (FMNH2), a long chain aliphatic aldehyde and $O_2$, to produce green-blue light. This enzyme can be readily classed as slow or fast decay based on their rate of luminescence decay in a single turnover. Mutation of Glu175 in $\alpha$ subunit to Gly converted slow decay Xenorhabdus Luminescence luciferase to fast decay one. The following studies revealed that changing the luciferase flexibility and lake of Glu-flavin interactions are responsible for the unusual kinetic properties of mutant enzyme. Optical and thermodynamics studies have caused a decrease in free energy and anisotropy of mutant enzyme. Moreover, the role of Glu175 in transition state of folding pathway by use of stopped-flow fluorescence technique has been studied which suggesting that Glu175 is not involved in transition state of folding and appears as surface residue of the nucleus or as a member of one of a few alternative folding nuclei. These results suggest that mutation of Glu175 to Gly extended the structure of Xenorhabdus Luminescence luciferase, locally.

Real Time Scale Measurement of Inorganic Phosphate Release by Fluorophore Labeled Phosphate Binding Protein (형광단이 붙어 있는 인산결합 단백질에 의한 인산 배출의 실시간 측정)

  • Jeong Yong-Joo
    • Journal of Life Science
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    • v.15 no.6
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    • pp.935-940
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    • 2005
  • Fluorescence change of coumarin labeled phosphate binding protein (PBP-MDCC) was monitored to measure the amount of released inorganic phosphate ($P_{i}$) during nucleoside triphosphate (NTP) hydrolysis reaction. After purification of PBP-MDCC, fluorescence emission spectra showed that fluorescence responded linearly to $P_{i}$ up to about 0.7 molar ratio of $P_{i}$ to protein. The correlation of fluorescence signal and $P_{i}$ standard was measured to obtain [$P_{i}$] - fluorescence intensity standard curve on the stopped-flow instrument. When T7 bacteriophage helicase, double-stranded DNA unwinding enzyme using dTTP hydrolysis as an energy source, reacted with dTTP, the change of fluorescence was able to be converted to the amount of released $P_{i}$ by the $P_{i}$ standard curve. $P_{i}$ release results showed that single-stranded Ml3 DNA stimulated dTTP hydrolysis reaction several folds by T7 helicase. Instead of end point assay in NTP hydrolysis reaction, real time $P_{i}$-release assay by PBP-MDCC was proven to be very easy and convenient to measure released $P_{i}$.

Folding Mechanism of WT* Ubiquitin Variant Studied by Stopped-flow Fluorescence Spectroscopy

  • Park, Soon-Ho
    • Bulletin of the Korean Chemical Society
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    • v.31 no.10
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    • pp.2877-2883
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    • 2010
  • The folding kinetics of $WT^*$ ubiquitin variant with valine to alanine mutation at sequence position 26 (HubWA) was studied by stopped-flow fluorescence spectroscopy. While unfolding kinetics showed a single exponential phase, refolding reaction showed three exponential phases. The semi-logarithmic plot of urea concentration vs. rate constant for the first phase showed v-shape pattern while the second phase showed v-shape with roll-over effect at low urea concentration. The rate constant and the amplitude of the third phase were constant throughout the urea concentrations, suggesting that this phase represents parallel process due to the configurational isomerization. Interestingly, the first and second phases appeared to be coupled since the amplitude of the second phase increased at the expense of the amplitude of the first phase in increasing urea concentrations. This observation together with the roll-over effect in the second folding phase indicates the presence of intermediate state during the folding reaction of HubWA. Quantitative analysis of Hub-WA folding kinetics indicated that this intermediate state is on the folding pathway. Folding kinetics measurement of a mutant HubWA with hydrophobic core residue mutation, Val to Ala at residue position 17, suggested that the intermediate state has significant amount of native interactions, supporting the interpretation that the intermediate is on the folding pathway. It is considered that HubWA is a useful model protein to study the contribution of residues to protein folding process using folding kinetics measurements in conjunction with protein engineering.

Transient Flow Instability inside a Gas Turbine Shaft (가스 터빈 축 내부의 비정상 유동의 불안정성)

  • Hur, Nahm-Keon;Won, Chan-Shik
    • The KSFM Journal of Fluid Machinery
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    • v.2 no.1
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    • pp.103-107
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    • 1999
  • Transient flow inside a hollow shaft of a Gas Turbine engine during sudden engine stop may result in non uniform heat transfer coefficients in the shaft due to flow instability similar to steady Taylor vortex, which may decrease the lifetime of the shaft. In the present study, transient Taylor vortex phenomena inside a suddenly stopped hollow shaft are studied analytically. Flow visualization is also performed to study the shape and onset time of Taylor Vortices for various initial rotational speed.

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Underground temperature survey for the study of shallow groundwater flow system

  • Okuyama Takehiko;Kuroda Seiichiro;Nakazato Hiroomi;Natsuka Isamu
    • 한국지구물리탐사학회:학술대회논문집
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    • pp.690-694
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    • 2003
  • Groundwater preferentially flows through sediment layers with high permeability such as colluvium. Its flow paths are called groundwater vein streams. An underground temperature survey is a method to locate vein streams by underground temperature anomalies associated with flowing groundwater. A groundwater flow system near an irrigation reservoir located in the upper part of a landslide block was surveyed with this method. After a geomembrane lining was installed in the reservoir, the total cross-sectional area of the vein streams in the aquifer decreased to as little as 0.35 times that before installation of the liner. A change in groundwater quality also indicated that the mixing of groundwater with leaked water from the reservoir stopped after installation of the lining.

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Effects of Nelumbinis Semen on Contractile Dysfunction in Ischemic and Reperfused Rat Heart

  • Kim, Jong-Hoon;Kang, Moon-Kyu;Cho, Chong-Woon;Chung, Hwan-Suck;Kang, Chang-Woon;Parvez, Shoukat;Bae, Hyun-Su
    • Archives of Pharmacal Research
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    • v.29 no.9
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    • pp.777-785
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    • 2006
  • Nelumbinis Semen (NS), or lotus seed, is one of the most well-known traditional herbal medicines and is frequently used to treat cardiovascular symptoms in Korea. The anti-ischemic effects of NS on ischemia-induced isolated rat heart were investigated through analyses of changes in blood pressure, aortic flow, coronary flow, and cardiac output. The subjects in this study were divided into two groups: a control, untreated ischemia-induced group, and an ischemia-induced group treated with NS. There were no significant differences in perfusion pressure, aortic flow, coronary flow and cardiac output between the groups before ischemia was induced. The supply of oxygen and buffer was stopped for ten minutes to induce ischemia in isolated rat hearts, and NS was administered during ischemia induction. NS treatment significantly prevented decreases in perfusion pressure, aortic flow, coronary flow and cardiac output under ischemic conditions (p<0.01). In addition, the mechanism of the anti-ischemic effects of NS was also examined through quantitation of intracellular calcium content in rat neonatal cardiomyocytes. NS significantly prevented intracellular calcium increases induced by isoproterenol (p<0.01). These results suggest that NS has distinct anti-ischemic effects through calcium antagonism.

Myocardial Protection of Contractile Function After Global Ischemia by Compound K in the Isolated Heart

  • Kim, Jong-Hoon
    • Journal of Ginseng Research
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    • v.33 no.4
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    • pp.268-277
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    • 2009
  • Ginsenosides are among the most well-known traditional herbal medicines frequently used for the treatment of cardiovascular symptoms in South Korea. The anti-ischemic effects of compound K (CK), a metabolite of ginsenoside Rb1, on ischemia-induced isolated rat hearts were investigated through the analyses of the changes in the hemodynamics (blood pressure, aortic flow, coronary flow, and cardiac output) and the measurement of the infarct region. The subjects in this study were divided into four groups: the normal control, the CK-alone group, the ischemia-induced group without any treatment, and the ischemia-induced group treated with CK. No significant differences in perfusion pressure, aortic flow, coronary flow, and cardiac output were found between the groups before ischemia was induced. The oxygen and buffer supply was stopped for 30 min to induce ischemia 60 min after reperfusion in the isolated rat hearts, and the CK was administered 5 min before ischemia induction. The CK treatment significantly prevented decreases in perfusion pressure, aortic flow, coronary flow, and cardiac output under ischemic conditions. In addition, the hemodynamics (except for the heart rate) of the group treated with CK significantly recovered 60 min after reperfusion, unlike in the control group. CK significantly limited the infarct. These results suggest that CK treatment has distinct anti-ischemic effects in an exvivo model of an ischemia-reperfusion-induced rat heart.