Kinetics of ligand binding to CYP3A4 was monitored at ambient temperatures in 0.1M phosphate, pH 7.4, in a SX.18MV stopped flow apparatus (Applied Photophysics, UK). After mixing 2 μM CYP3A4 with a 20 μM ligand solution, absorbance changes at 427 nm were measured to follow conversion of the heme iron to the low-spin form. Kinetic data were analyzed using manufacturer’s PROKIN software.
Sx 18mv stopped flow apparatus
Manufactured by Applied Photophysics
Sourced in United Kingdom
The SX.18MV stopped flow apparatus is a laboratory instrument designed for the rapid mixing and analysis of chemical and biological samples. It is capable of measuring rapid reactions with time scales as short as 1 millisecond. The instrument consists of a drive unit, a mixer, and a detector module that can accommodate various types of detectors such as UV-Visible, fluorescence, and circular dichroism.
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12 protocols using sx 18mv stopped flow apparatus
1
Kinetics of CYP3A4 Ligand Binding
2
Stopped-flow Kinetic Measurements of Ca2+ Chelation
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All kinetic measurements were carried out in stopped-flow buffer (10 mM MOPS, 150 mM KCl, pH 7.0) at 15°C using an Applied Photophysics Ltd. (Leatherhead, UK) model SX.18MV stopped-flow apparatus with a dead time of ~1.4 ms. IAANS fluorescence was excited at 330 nm and monitored using a 420–470 nm band-pass interference filter (Oriel, Stratford, CT). Ca2+ chelator EGTA (10 mM) in stopped-flow buffer was used to remove Ca2+ (200 μM) from the chimera (0.5 μM) also in stopped-flow buffer in the absence or presence of compounds. Varying concentration of each compound were individually added to both stopped-flow reactants. Data traces were fit using a program by P.J. King (Applied Photophysics, Ltd.), that utilizes the non-linear Lavenberg-Marquardt algorithm. Each koff represents an average of at least three separate experiments ± standard error, each averaging at least three shots fit with a single exponential equation.
3
Anaerobic Kinetic Measurement of Cytochrome b5 Reduction
4
Monitoring Ribosomal Subunit Association by Light Scattering
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The association of ribosomal subunits was monitored by light scattering after rapid mixing in an SX.18MV stopped-flow apparatus (Applied Photophysics, Leatherhead, UK), as previously described (33 (link),60 (link),61 (link)). The excitation wavelength was 435 nm (4.65 nm bandpass) and the scattered light was measured at an angle of 90° to the incident beam. Before mixing, the 30S and 50S subunits were incubated at least 30 min at 51°C. Two mixes were prepared as follows. The mix corresponding to Syringe 1 contained 30S ribosomal subunits (50 nM), GTP (250 μM) with or without, depending on the experiment, mRNA (50nM), Met-tRNAiMet (50nM), aIF2-GTP (50nM), aIF1 (50nM) and aIF1A (50nM) (Table 2 ). Mix corresponding to Syringe 2 contained 50S ribosomal subunits (50 nM), GTP (250 μM) with or without, depending on the experiment, aIF5B or its variants (1 μM). In Table 2 , lane 8, GDPNP (250 μM) was used instead of GTP in syringe 2. Association was measured at 51°C after at least 30 min of incubation to complete hydrolysis of GTP bound to aIF2 in Syringe 1 (see Results). The observed association rate constants were estimated by non-linear least-square fitting of the scattered intensity (I) data points to a hyperbolic equation (I = Ifinal – D(1/1 + kobs*t)) as described (62 (link)). The Origin (OriginLab) software was used for fitting (Figure 1B ).
5
Oxygen Complex Formation and Decay
6
Kinetics of Ligand Binding to CYP3A4
7
Measuring Ligand Binding Kinetics in CYP3A4
8
Kinetics of Ca2+ Dissociation from T53C-IAANS
9
Kinetics of CYP3A4 Ligand Binding
10
Stopped-Flow Kinetics of Compound I Formation
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Pre-steady-state spectroscopic
changes induced by addition of H2O2 were measured
using a SX-18MV stopped-flow apparatus equipped with a diode array
detector from Applied Photophysics. The optical quartz cell with a
path length of 10 mm had a volume of 20 μL. The fastest time
for mixing was 1 ms, and all measurements were performed at 25 °C.
The ferric protein (2 μM) in 50 mM phosphate buffer (pH 7.0)
was mixed with either 2 or 25 μM H2O2 and
measured in triplicate for 10 and 100 ms. Second-order rate constants
(kapp) for the formation of Compound I
were calculated using ProK IV global fitting software assuming a pseudo-first-order
reaction and the model A + B > C.
changes induced by addition of H2O2 were measured
using a SX-18MV stopped-flow apparatus equipped with a diode array
detector from Applied Photophysics. The optical quartz cell with a
path length of 10 mm had a volume of 20 μL. The fastest time
for mixing was 1 ms, and all measurements were performed at 25 °C.
The ferric protein (2 μM) in 50 mM phosphate buffer (pH 7.0)
was mixed with either 2 or 25 μM H2O2 and
measured in triplicate for 10 and 100 ms. Second-order rate constants
(kapp) for the formation of Compound I
were calculated using ProK IV global fitting software assuming a pseudo-first-order
reaction and the model A + B > C.
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