Lambda 45 uv vis spectrophotometer

The structural characterization of the films was performed by X-ray diffraction measurements performed with a Bruker D8 Advance X-ray diffractometer (Bruker AXS GmbH, Karlsruhe, Germany) using CuKα radiation (λ = 1.54178 Å) in the 20–70° range. The surface morphology of ZnO nanostructures/Cu and ZnO/Graphene/Cu was analyzed using a Gemini 500 field emission scanning electron microscope from Zeiss (Oberkochen, Germany). The Raman spectra of the ZnO/Cu and ZnO/G/Cu films were recorded at room temperature using a LabRAM HR Evolution Raman spectrometer (Horiba Jobin-Yvon: Edison, NJ, USA), with a He–Ne laser functioning at 633 nm being focused by an Olympus 100× objective on the surface of the films. The reflectance spectra were recorded using a PerkinElmer Lambda 45 UV–Vis spectrophotometer(Waltham, Massachusetts, USA) and the photoluminescence measurements were performed using an Edinburgh FL 920 PL spectrometer (Edinburgh Instruments, Livingston, UK), with 325 nm UV excitation at room temperature.

The catalytic wet hydrogen peroxide oxidation (CWHPO) reactions were carried out in a five-necked glass reactor thermostated at 25 °C under continuous stirring (300 rpm) and purging with nitrogen. In a typical run, 250 mL aqueous solution of dye (MB, PC, RZ) with a concentration of 25 mg/L was transferred into the reactor followed by adding 100 mg of the catalyst. The reaction mixture was kept under these conditions for 60 min for adsorption-desorption equilibrium to be established. After that, the oxidizing agent (H₂O₂) with a concentration of 3.3% was added. At appropriate time intervals (up to 300 min), 1 mL aliquots were withdrawn and immediately centrifuged at 6000 rpm for 2 min for the removal of catalyst particles. The temporal concentration of dyes was determined using a Lambda 45 UV-Vis spectrophotometer (Perkin Elmer Inc, Waltham, MA, USA) measuring absorbance at 664 nm (MB), 550 nm (PC) and 600 nm (RZ). The catalytic activity of the examined materials in all reactions was tested in triplicate to confirm the reproducibility of results.

The absorption spectra of samples and references were plotted in the 250–500 nm range, on a Perkin-Elmer Lambda 45 UV-Vis spectrophotometer equipped with a thermostated cell holder at 25.0 ± 0.1°C. Derivative spectra were made from absorption spectra after subtracting the absorption of the references and using an Excel® based routine previously published (Magalhães et al., 2010 (link)).
The fluorescence emission spectra of samples and references were obtained in the 300–500 nm range, at a scanning rate of 50 nm.min⁻¹, during 10 s integration time on a Perkin-Elmer LS-50B spectrofluorimeter equipped with a thermostated cell holder at 25.0 ± 0.1°C and λ_excitation = 272 nm. Three independent experiments were carried out. Derivative spectra were made from fluorescence emission spectra using Microcal Origin 9.0® software further used for data treatment.

Scanning electron microscope (SEM) images were acquired on a Merlin Compact (Zeiss, Oberkochen, Germany) with an accelerating voltage of 10 kV. Transmission electron microscopy (TEM) images were acquired on a FEI Talos F200c (ThermoFisher, Waltham, MA, USA) at 120 kV. UV-Vis spectra were acquired using a Lambda 45 UV-Vis spectrophotometer (PerkinElmer, Waltham, MA, USA). Fourier transform infrared spectra (FTIR) were acquired using a VERTEX V70 Fourier infrared spectrometer (Bruker, Karlsruhe, Germany). Raman spectra were recorded with a laser confocal Raman spectrometer (XperRam200, Nanobase, Seoul, Korea) equipped with an Olympus upright microscope with a 40× objective, numerical aperture (NA) of 0.65, and an Andor scientific grade TE-cooled CCD. The Raman excitation wavelength was set to 785 nm, the power was 60 mW, the integration time was set to 1 s, and the spectral resolution was 2.5 cm⁻¹. All Raman-testing experiments were performed on silicon substrates. The baseline of spectral data was removed by the LabSpec software (Horiba, Paris, France).

h12-LOX reactions were performed at 22, 37, and 42 °C in a 1 cm² quartz cuvette containing 2 mL of 25 mM HEPES (pH 7.5) with substrate (docosahexaenoic acid (DHA) and AA). DHA and AA concentrations were varied from 0.25 to 10 μM. Concentrations of DHA and AA were determined by measuring the amount of oxylipins produced from complete reaction with soybean lipoxygenase-1 (SLO-1). Concentrations of oxylipins were determined by measuring the absorbance at 234 nm. Reactions were initiated by the addition ~20 μg of WT and ~30 μg of Y649C h12-LOX and were monitored on a PerkinElmer Lambda 45 UV/Vis spectrophotometer. Product formation was determined by the increase in absorbance at 234 nm for oxylipin products (ε_234nm = 25,000 M⁻¹ cm⁻¹), within the first 20 s of the reaction where no enzyme inactivation was observed. KaleidaGraph (Synergy) was used to fit initial rates (at less than 20% turnover), to the Michaelis-Menten equation for the calculation of kinetic parameters. For the stability assay, all reactions were conducted using a Cary-UV Vis spectrophotometer, which was set to 22 and 37 °C, with 25 mM HEPES buffer (pH 8) and stirred continuously. Stocks of WT and Y649C at 133 nM and 200 nM respectively were incubated at 22 and 37 °C, with 6000 μL being withdrawn at each time point. The fastest rates over 15 s intervals were plotted versus the enzyme incubation time at 37 °C.

Overexpression and purification of human 12‐LOX were performed as previously described.¹⁷ 12‐LOX steady‐state kinetic reactions were constantly stirred at ambient temperature, in a 1 cm² quartz cuvette containing 2 ml of 25 mM HEPES, pH 8 with DGLA, AA, EPA, 15‐HpETrE, 15‐HpETE, or 15‐HpEPE. Substrate concentrations were varied from 0.25 to 10 μM for the PUFA reactions or 0.5 to 25 μM for the 15‐oxylipin reactions. Concentrations of PUFA were determined by measuring the amount of 15‐oxylipin produced from a complete reaction with soybean lipoxygenase‐1. Concentrations of 5S‐HETE, 5S‐HpETE, 7S‐HDHA, and 7S‐HpDHA were determined by measuring the absorbance at 234 nm. Reactions were initiated by the addition of h15‐LOX‐1 (~20‐60 nmol) and were monitored on a Perkin‐Elmer Lambda 45 UV/VIS spectrophotometer. Product formation was determined by the increase in absorbance at 234 nm for 15‐oxylipins (ε₂₃₄ = 27 000 M⁻¹ cm⁻¹) and 270 nm for di‐oxylipins (ε₂₇₀ = 37 000 M⁻¹ cm⁻¹).¹⁸, ¹⁹ KaleidaGraph (Synergy) was used to fit initial rates, as well as the second‐order derivatives (k_cat/K_M) to the Michaelis‐Menten equation for the calculation of kinetic parameters.