To study the effect of pH on PmPV1 structural stability, the protein was incubated overnight in different buffers ranging from pH 2.0 to 12.0 following a previously reported method24 (link). Samples were analysed by absorbance and fluorescence spectroscopy and by SAXS. Absorbance spectra of were recorded between 300–650 nm in an Agilent 8453 UV/Vis diode array spectrophotometer (Agilent Technologies, Waldbronn, Germany) taking advantage of the fact that PmPV1 is a carotenoprotein and the protein-carotenoid interaction could be followed by its spectrum in this range. Fluorescence emission was recorded as described in the Chemical denaturation section. Two independent samples were measured three times and the corresponding buffer blank was subtracted. The effect of temperature on PmPV1 at pH 7.4 was also measured by absorption and fluorescence spectroscopy and by SAXS in the range 25–85 °C. The effect of extreme thermal conditions was analysed by boiling PmPV1 for 50 min and evaluating the oligomer integrity using native (non-denaturing) gel electrophoresis and the carotenoprotein fine spectra using absorbance spectrophotometry.
Agilent 8453 uv vis diode array spectrophotometer
Manufactured by Agilent Technologies
Sourced in Germany
The Agilent 8453 UV/Vis diode array spectrophotometer is a high-performance instrument used for quantitative and qualitative analysis of various samples. It employs a diode array detector to simultaneously measure the entire UV-visible spectrum, providing rapid and efficient data collection. The instrument is designed to deliver accurate, reliable, and reproducible results across a wide range of applications.
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5 protocols using agilent 8453 uv vis diode array spectrophotometer
1
Structural Stability of PmPV1 Protein
2
Spectrophotometric Determination of Host-Guest Binding
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UV–vis absorption spectra were recorded on an Agilent 8453 UV–vis diode array spectrophotometer (200−800 nm; Agilent Technologies, Waldbronn, Germany), using 1 cm path-length quartz cuvettes. Changes in the absorption intensity of 1a (λ = 240 nm) were monitored as a function of β-CD or HP- β-CD concentration to determine the binding constant (Kb), which was calculated by non-linear squares fitting with OriginPro 8.5.1 (Northampton, MA, USA). In these experiments the concentration of 1a was kept constant at 2.2 × 10−4 M, whereas the concentration of β-CD and HP-β-CD varied from 0 to 6.9 × 10−3 M (1:30 molar ratio) by direct titration with a micropipette on the measuring cell.
3
Carotenoproteins Absorption Spectra in Pomacea
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Absorption spectra of egg carotenoproteins are valuable taxonomic characters in Pomacea spp. (Pasquevich & Heras, 2020 (link)). PV1s absorb light in the visible region of the spectrum because of the presence of carotenoids pigments (Heras et al., 2007 ). Absorption spectra of PVF and purified carotenoproteins were recorded between 350 nm to 650 nm in an Agilent 8453 UV/Vis diode array spectrophotometer (Agilent Technologies, Waldbronn, Germany).
4
Nanomaterials Characterization by Advanced Techniques
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Transmission electron microscope (TEM) Image measurements of nanomaterials were carried out on a JEM-2000EX II (JEOL, Akishima, Tokyo, Japan) at 100 kV. Photoluminescence spectra were measured by FSP-920 (Edinburgh Instruments, Livingston, West-Lothian, Scotland) spectrometer equipped with a 793-nm diode laser. The UV–Vis absorption spectra were measured by Agilent 8453 diode array UV–Vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). The dynamic light scattering (DLS) measurement was determined by SZ-100 zetasizer (HORIBA Scientific, Kyoto, Kyoto Prefecture, Japan). Cell imaging was performed on an FV1000-IX81 laser confocal microscope (Olympus, Shinjuku, Tokyo, Japan).
5
Dissolution Kinetics of Xan Tablets
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To determine drug release from the Xan tablets, the collected samples of the outflow medium from the MRI flow-through cell were filtered through a filter with 0.45 μm pores. Drug release was monitored as a function of time using the HP Agilent 8453 Diode Array UV-Vis Spectrophotometer, Waldbronn, Germany, to measure the absorbance of PF at 274 nm.
Drug release profiles were analyzed using the Korsmeyer–Peppas equation [41 (link),42 (link)]:
where M(t)/M(∞) is the fraction of the released drug at time t, k is the rate constant, and n is the diffusion exponent, which indicates the general release mechanism: n = 0.5 indicates Fickian diffusion controlled drug release (Case I), and n = 1.0 indicates Case II transport (erosion controlled drug release). Case I release occurs by molecular diffusion of the drug due to a chemical potential gradient, whereas the mechanism driving Case II drug release is the swelling or relaxation of polymeric chains. Values between 0.5 < n < 1.0 indicate an anomalous (non-Fickian or both diffusion/erosion) controlled drug release. Equation (1) is the short time approximation, which is valid only up to the fraction of 0.6 of released drug.
Drug release profiles were analyzed using the Korsmeyer–Peppas equation [41 (link),42 (link)]:
where M(t)/M(∞) is the fraction of the released drug at time t, k is the rate constant, and n is the diffusion exponent, which indicates the general release mechanism: n = 0.5 indicates Fickian diffusion controlled drug release (Case I), and n = 1.0 indicates Case II transport (erosion controlled drug release). Case I release occurs by molecular diffusion of the drug due to a chemical potential gradient, whereas the mechanism driving Case II drug release is the swelling or relaxation of polymeric chains. Values between 0.5 < n < 1.0 indicate an anomalous (non-Fickian or both diffusion/erosion) controlled drug release. Equation (1) is the short time approximation, which is valid only up to the fraction of 0.6 of released drug.
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