One-point inhibition percentages of approximately 100 derivatives of MLS000099089 (99089 ), the parent compound, were determined by following the formation of the conjugated diene product at 234 nm (ε = 27,000 M−1cm−1) with a Perkin-Elmer Lambda 40 UV/Vis spectrophotometer at 20 µM inhibitor concentration. Twenty-nine selective derivatives were investigated further to determine their IC50 values. The full IC50 experiments were done with at least four different inhibitor concentrations. All reactions mixtures were 2 mL in volume and constantly stirred using a magnetic stir bar at room temperature (23°C) with the appropriate amount of LOX isozyme h12/15-LOX (~ 30 nM). Reactions were carried out in 25 mM HEPES buffer (pH 7.5), 0.01% Triton X-100 and 10 µM AA. The concentration of AA was quantitated by allowing the enzymatic reaction to proceed to completion in the presence of s12/15-LOX. IC50 values were obtained by determining the enzymatic rate at various inhibitor concentrations and plotted against inhibitor concentration, followed by a hyperbolic saturation curve fit. The data used for the saturation curves were performed in duplicate or triplicate, depending on the quality of the data. All inhibitors were stored at −20°C in DMSO.
Lambda 40 uv vis spectrophotometer
Manufactured by PerkinElmer
Sourced in United States, United Kingdom, Italy
The Lambda 40 UV/Vis spectrophotometer is a compact and versatile instrument designed to measure the absorbance or transmittance of light in the ultraviolet and visible regions of the electromagnetic spectrum. It features a dual-beam optical system and a monochromator to provide accurate and reliable measurements across a wide wavelength range.
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Lab products found in correlation
Kieselgel 60, by Merck Group (2 mentions)
Kieselgel 60 f254 aluminum plates, by Merck Group (2 mentions)
Alpha 2 ft ir spectrometer, by Bruker (2 mentions)
Kaleidagraph, by Synergy Software (2 mentions)
Drx 400, by Bruker (1 mentions)
Ltq orbitrap velos mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Avance 3 600, by Bruker (1 mentions)
Avance neo 700, by Bruker (1 mentions)
Drx 400 spectrometer, by Bruker (1 mentions)
Dsq mass spectrometer, by Thermo Fisher Scientific (1 mentions)
31 protocols using lambda 40 uv vis spectrophotometer
1
Inhibition of 12/15-LOX by MLS000099089 Derivatives
2
Fluconazole Solubility Enhancement by HP-β-CD
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Phase solubility studies were carried out according to the method reported by Higuchi and Connors [23 ]. Excess amounts of fluconazole were added to 10 mL of distilled water containing increasing concentrations of HP- -CD, specifically 0.071, 1.43, 2.14, 2.86, 5.72 and 9.23 mM of cyclodextrin. The resulting dispersions were maintained under magnetic stirring at 37.0 ± 0.1 °C for 72 h. After this time, the suspensions were left to settle, then 1 mL of supernatant was taken and appropriately diluted with distilled water (1:10), taking care to not alter temperature. Fluconazole concentration was determined by measuring the UV absorption at 260 nm and 37.0 ± 0.1 °C with a Perkin Elmer Lambda 40 UV-Vis spectrophotometer. Calibration curve for fluconazole reference standard was obtained by measuring the UV absorption ( = 260 nm) in an ethanol/water 50:50 (v/v) solution at 37.0 ± 0.1 °C. The linearity of the calibration curve was confirmed in the range 0.066–1.32 mg/mL with a regression coefficient (R ) value of 0.995 [24 (link),25 ,26 ].
3
β-Carotene Bleaching Antioxidant Assay
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The β-carotene bleaching test was used to evaluate the antioxidant activities based on the β-carotene lenolate system model [18 ], with slight modifications. Briefly, 1 mL of β-carotene solution (0.2 mg/mL in chloroform) was added to 0.02 mL of linoleic acid and 0.2 mL of 100 % Tween 20. Then, 5 mL samples of this emulsion were transferred into test tubes containing 0.2 mL of test samples in 80 % methanol at increasing concentrations (62.5, 125, 250, 500, and 1000 μg/mL). These mixtures were then incubated in a water bath at 40 °C for 120 min. All determinations were performed in duplicate and the mean values calculated. The absorbance was measured at 470 nm using a Perkin–Elmer Lambda 40 UV/Vis spectrophotometer against a blank consisting of the emulsion without β-carotene. The measurements were carried out at initial and final times (t = 0 and 120 min, respectively) with propyl gallate as the positive control. The AA% was measured and expressed as the percentage of inhibition of β-carotene oxidation using the following equation:
Where AS0 and AS120 are absorbance values of the samples and AC0, and AC120 are the controls at 0 and 120 min, respectively.
Where AS0 and AS120 are absorbance values of the samples and AC0, and AC120 are the controls at 0 and 120 min, respectively.
4
Thermal Melting Curve Analysis of Gapmer-RNA Duplexes
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Equal amounts of gapmer and RNA were dissolved in buffer (10 mM phosphate buffer, 100 mM NaCl, 0.1 nM ethylenediaminetetraacetic acid (EDTA), pH 7.0) to final concentrations of 1.5 mM for each. Samples were denatured at 95°C for 3 min and then allowed to anneal by slowly cooling to room temperature for 30 min. Thermal melting curves were recorded at 260 nm on a Lambda 40 UV/VIS Spectrophotometer equipped with a PTP6 Peltier Temperature Programmer (Perkin Elmer, Waltham, USA) using a temperature gradient that was increased by 1°C/min from 20°C to 95°C and then decreased to 58°C. First derivatives and the local maxima of both the melting and annealing were used to assess the duplex melting temperature (Tm), defined as the temperature at which half of the gapmers are duplexed with RNA.
5
Quantification of Total Flavonoids
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Total flavonoid content was quantified according to the protocol described by Kong et al. [31 (link)]. Briefly, 100 µL of plant extract, 150 µL of sodium nitrite (5%), and 150 µL of aluminum trichloride (10%) were mixed. Then, after 5 min, 200 µL of hydroxide sodium (1%) was added to the mixture. The mixture was vortexed and incubated for one hour. The absorbance was measured at 510 nm using the Perkin Elmer Lambda 40 UV/VIS spectrophotometer. The results obtained were expressed as mg equivalent quercetin per gram of extract (mg QE/g).
6
Inhibition of 5-Lipoxygenase Activity
7
Spectroscopic Characterization of Organic Compounds
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Optical rotations were measured on a Krüss polarimeter (A. KRÜSS Optronic GmbH, Hamburg, Germany) equipped with a 0.5 dm cell. UV spectra were recorded on a Lambda 40 UV/Vis spectrophotometer (Perkin Elmer Ltd., Beaconsfield, UK). IR spectra were obtained on a Alpha II FTIR spectrometer (Bruker Optik GmbH, Ettlingen, Germany). High-resolution APCI mass spectra were measured on a LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). NMR spectra were recorded on Avance NEO 950, Avance NEO 700, Avance III 600, and DRX 400 spectrometers (Bruker BioSpin GmbH, Rheinstetten, Germany). Chemical shifts are given on a δ (ppm) scale using TMS as internal standard. The 2D experiments (HSQC, HMBC, COSY, NOESY) were performed using standard Bruker pulse sequences. Column chromatography separations were performed with Kieselgel 60 (Merck, Darmstadt, Germany). HPLC separations were conducted using a Waters 600 liquid chromatography pump equipped with a Waters 410 differential refractometer (Waters Corporation, Milford, MA, USA), using a 25 cm × 10 mm Econosphere Silica 10 μ column (Grace, Columbia, MD, USA). TLC were performed with Kieselgel 60 F254 aluminum plates (Merck, Darmstadt, Germany) and spots were detected after spraying with 20% H2SO4 in MeOH reagent and heating at 100 °C for 1 min.
8
Spectroscopic Characterization of Compounds
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Optical rotations were measured on a Krüss polarimeter (A. KRÜSS Optronic GmbH, Hamburg, Germany) equipped with a 0.5 dm cell. UV spectra were recorded on a Lambda 40 UV/Vis spectrophotometer (Perkin Elmer Ltd., Beaconsfield, UK). IR spectra were obtained on an Alpha II FTIR spectrometer (Bruker Optik GmbH, Ettlingen, Germany). Low-resolution EI mass spectra were measured on a Thermo Electron Corporation DSQ mass spectrometer (Thermo Electron Corporation, Austin, TX, USA) using a Direct-Exposure Probe (Thermo Electron Corporation, Austin, TX, USA). NMR spectra were recorded on a DRX 400 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany). The 2D experiments (HSQC, HMBC, COSY, NOESY) were performed using standard Bruker pulse sequences. Column chromatography separations were performed with Kieselgel 60 (Merck, Darmstadt, Germany). HPLC separations were conducted on a Pharmacia LKB 2248 liquid chromatography pump (Pharmacia LKB Biotechnology, Uppsala, Sweden) equipped with a RI-102 Shodex refractive index detector (ECOM spol. s r.o., Prague, Czech Republic) using an Econosphere Silica 10 μm (250 × 10 mm i.d.; Grace, Columbia, MD, USA) column. TLC were performed with Kieselgel 60 F254 aluminum plates (Merck, Darmstadt, Germany) and spots were detected after spraying with 20% H2SO4 in MeOH reagent and heating at 100 °C for 1 min.
9
Kinetic analysis of hALOX12 mutants
10
Kinetic Analysis of 12-LOX Inhibition
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