Pre-steady-state kinetics were studied by the stopped-flow technique using an SX20 stopped-flow spectrometer (Applied Photophysics Ltd., Leatherhead, United Kingdom). The fluorescence of FAM was excited at 494 nm, and the Förster resonance energy transfer (FRET) signal was monitored at wavelengths ≥530 nm by means of an OG-530 filter (Schott, Mainz, Germany). Trp was excited at 290 nm, and its fluorescence emission was monitored at wavelengths ≥320 nm using a WG-320 filter (Schott, Mainz, Germany). 2-Aminopurine (aPu) fluorescence was excited at 310 nm, and its emission was monitored at wavelengths ≥370 nm with an LG-370 Corion filter. The dead time of the instrument is 1.4 ms. Typically, each trace shown is an average of three or more individual experiments. Experimental error was less than 5%. Experiments with α-A-, ε-A-, DHU-, and U-containing DNA substrates were conducted in NIR buffer. Experiments with the F-site-containing DNA (F-substrate) and a nondamaged DNA duplex were performed in BER buffer. The solutions containing the enzyme and substrate were loaded into two separate syringes of the stopped-flow instrument and were incubated for an additional 3 min at 25°C prior to mixing. The reported concentrations of reactants are those in the reaction chamber after mixing.
Sx20 stopped flow spectrometer
Manufactured by Applied Photophysics
Sourced in United Kingdom
The SX20 stopped-flow spectrometer is a laboratory instrument designed for the rapid mixing and analysis of chemical or biological samples. It is capable of acquiring kinetic data at millisecond time scales, enabling the study of fast reactions and processes.
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Lab products found in correlation
Cary 100 bio uv vis spectrometer, by Agilent Technologies (2 mentions)
Wg 320 filter, by Schott (1 mentions)
Varian cary 50 spectrophotometer, by Agilent Technologies (1 mentions)
Varioskan flash plate reader, by Thermo Fisher Scientific (1 mentions)
Rx2000 rapid mixing stopped flow unit, by Applied Photophysics (1 mentions)
Sigma plot v 7, by Merck Group (1 mentions)
Ph meter basic 20, by Crison (1 mentions)
Gg 435, by Schott (1 mentions)
Micro bio spin 6 column, by Bio-Rad (1 mentions)
Infinite m1000 pro, by Tecan (1 mentions)
Kaleidagraph program, by Synergy Software (1 mentions)
Anaerobic chamber, by Coy Laboratory Products (1 mentions)
Og 515, by Schott (1 mentions)
Vapor pressure osmometer, by Wescor (1 mentions)
Pro data viewer, by Applied Photophysics (1 mentions)
Esr900 helium flow cryostat, by Oxford Instruments (1 mentions)
Breeze 2 system, by Waters Corporation (1 mentions)
E680 spectrometer, by Bruker (1 mentions)
Bovine catalase, by Merck Group (1 mentions)
D glucose, by Merck Group (1 mentions)
42 protocols using sx20 stopped flow spectrometer
1
Stopped-Flow Kinetics of FRET and Fluorescence
2
Kinetics of RK-GB1 Binding to Recoverin
3
Kinetic Analysis of Menadione Reductase
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All kinetic analyses were performed in 50 mM Tris-Cl, 150 mM NaCl, 1% octylglucoside (OG), pH 8.0. The OG provides a hydrophobic volume to accommodate the menadione substrate and maintains the enzyme in an active state. Steady-state kinetic measurements were at 37 °C in a 96-well SpectraMax plate reader and monitored by the absorbance of NADH (340–380 nm, ε = 4.81 mM−1 cm−1) or APADH (400–450 nm, ε = 3.16 mM−1 cm−1). Enzyme concentrations used were typically 8 ng mL−1 for the wild-type reacting with NADH, but were increased up to 1 μg mL−1 for variants/reactions with lower turnover rates. Pre-steady-state measurements were at 23 °C in an SX20 stopped flow spectrometer (Applied Photophysics, dead time ~1 ms) fitted with a photodiode array detector and housed in an anaerobic glovebox. The flavin oxidation state was monitored by the average absorbance over 438 to 464 nm, and the CTC by the average over 639 to 684 nm (taking average values over these wavelength ranges improved the signal:noise ratio). When required, the flavin was pre-reduced using small aliquots of sodium dithionite, NADH or NADPH, then the concentration of reduced enzyme confirmed spectroscopically in control stopped-flow experiments against buffer-only solutions.
4
Kinetic Study of Iridium(III) Chloride Reduction
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A stock solution of 1.0 mM [IrCl6]2− was prepared and used daily by dissolving the desired amount of Na2IrCl6·6H2O in a solution mixture containing 0.99 M NaClO4 and 0.01 M HCl. Stock solutions of BH/PAH were prepared by adding the required amount of BH/PAH into a reaction medium of specific pH and then flushed for 5 min with nitrogen of high purity. For kinetic measurements, solutions of [IrCl6]2− and BH/PAH were prepared by dilution of the stock solutions with the same medium and then flushed for about 5 min with the nitrogen. Reactions were initiated by mixing equal volumes of the [IrCl6]2− and BH/PAH solutions directly on an SX-20 stopped-flow spectrometer (Applied Photophysics Ltd., Leatherhead, UK); the temperature was also controlled to ±0.1 °C using another thermostat of Lauda Alpha RA8. Moreover, the reaction solutions were only used for a couple of hours. The reactions were investigated under pseudo first-order conditions with [Hydrazide]tot ≥ 10·[IrCl62−].
5
Investigating α-Synuclein-Copper Redox Dynamics
6
Gramicidin-Based Fluorescence Assay for Phytochemical Bilayer Modulation
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The phytochemicals’
bilayer-modifying potency was determined using a gramicidin-based
fluorescence assay as described previously.42 (link),81 (link) Fluorophore-loaded large unilamellar lipid vesicles (LUVs) were
made of 1,2-dierucoyl-sn-glycero-3-phosphocholine
(DC22:1PC) using a mixture of hydration, sonication, freeze–thawing,
and extrusion. We use long-chain lipids to increase the bilayer thickness
sufficiently to shift the gramicidin monomer↔dimer equilibrium
toward the nonconducting monomers, which is necessary in order to
detect changes in the monomer↔dimer equilibrium. The LUVs were
doped with 260 nM gramicidin (gA) from Bacillus brevis 24 h before use and incubated at 12 °C in the dark. The phytochemicals
were incubated for 10 min at 25 °C with the LUV suspension. The
vesicle-entrapped fluorophore, 8-aminonaphthalene-1,3,6-trisulfonic
acid (ANTS), is quenched by a gramicidin permeable quencher (Tl+). The time course of fluorescence quenching was measured
using a SX.20 Stopped-Flow Spectrometer from Applied Photophysics,
excitation was set at 352 nm and the fluorescence emission above 455
nm was recorded. The quencher fluorescence was normalized to the initial
buffer value and a stretched exponential82 was fit to the first 2–100 ms and the rate at 2 ms was calculated.
Each phytochemical was measured in four to six experiments from at
least two different vesicle preparations.
bilayer-modifying potency was determined using a gramicidin-based
fluorescence assay as described previously.42 (link),81 (link) Fluorophore-loaded large unilamellar lipid vesicles (LUVs) were
made of 1,2-dierucoyl-sn-glycero-3-phosphocholine
(DC22:1PC) using a mixture of hydration, sonication, freeze–thawing,
and extrusion. We use long-chain lipids to increase the bilayer thickness
sufficiently to shift the gramicidin monomer↔dimer equilibrium
toward the nonconducting monomers, which is necessary in order to
detect changes in the monomer↔dimer equilibrium. The LUVs were
doped with 260 nM gramicidin (gA) from Bacillus brevis 24 h before use and incubated at 12 °C in the dark. The phytochemicals
were incubated for 10 min at 25 °C with the LUV suspension. The
vesicle-entrapped fluorophore, 8-aminonaphthalene-1,3,6-trisulfonic
acid (ANTS), is quenched by a gramicidin permeable quencher (Tl+). The time course of fluorescence quenching was measured
using a SX.20 Stopped-Flow Spectrometer from Applied Photophysics,
excitation was set at 352 nm and the fluorescence emission above 455
nm was recorded. The quencher fluorescence was normalized to the initial
buffer value and a stretched exponential82 was fit to the first 2–100 ms and the rate at 2 ms was calculated.
Each phytochemical was measured in four to six experiments from at
least two different vesicle preparations.
7
Rapid Kinetic Analysis of Thrombin-PPACK Binding
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Rapid kinetic experiments of PPACK binding were conducted on an Applied Photophysics SX20 stopped-flow spectrometer using an excitation of 295 nm and a cutoff filter at 320 nm. The dead time of the mixing cell for this instrument is 0.5–1 ms. Final concentrations of 150–250 nM thrombin wild-type or mutants were used in buffer containing 50 mM Tris, 0.1% PEG8000, 400 mM ChCl, pH 8.0, at 15 °C. The solution containing the protein was mixed 1:1 with 60 µL solutions of PPACK in the same buffer. Baselines were measured by mixing the protein into buffer in the absence of ligand. Each kinetic trace was taken as the average of at least ten determinations and fit to single or double exponentials based on the analysis of residuals using software supplied by Applied Photophysics. Values of the relaxations for single and double exponential fits were derived from at least three independent titrations.
8
Measuring Osmotic Permeability of Cells
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Water and glycerol permeability were measured using an SX20 stopped-flow spectrometer (Applied Photophysics). The BM-neutrophils were subjected to a 150-mM inwardly directed mannitol or glycerol gradient. The kinetics of the decreasing cell volumes was measured from the time course of 90 scattered light intensity at 450 nm wavelength. Osmotic water/glycerol permeability coefficients (Pf) were calculated as described previously35 (link).
9
Kinetics of Nitroalkene-Thiol Reactions
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The reaction between the nitroalkenes and the thiols βME and GSH was followed through the change in UV absorption of the nitroalkene, at 350 nm for NATOH and NATxME or 260 nm for NATx0. The fastest reactions were monitored in a SX20 stopped-flow spectrometer (Applied Photophysics); intermediate reactions (t > 10 s) were followed in a Varian Cary50 spectrophotometer (Agilent) using a RX2000 rapid mixing stopped-flow unit (Applied Photophysics); and the slowest reactions (t > 600 s) were studied using a Varioskan Flash plate reader (Thermo). The reactions were performed in TMA20 buffer at the specified pH and 25 °C. The time courses were fitted to a single exponential function (equation 1 ) and the resulting rate constants (kobs) were plotted vs the thiol concentration to obtain the rate constants of addition (kf) and elimination (kr).
10
Stopped-Flow Kinetics Analysis Protocol
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