Fluorescence spectrophotometer

A quarter of each filter was ultrasonically extracted in 10 mL of ultra-pure water for 20 min. Insoluble particles were removed by filtering each aqueous extract through a Teflon (PTFE) filter with a pore size of 0.45 μm using a syringe. A standard solution for OH radical generation was prepared by mixing pure water with 0.2, 0.4, 0.6, 0.8, and 1 mL of diluted hydroxyterephthalic acid (OHTA) solution. A mixture of 2 mL of diluted dithiothreitol (DTT) solution, 4 mL of sample extract, and 2 mL of disodium terephthalate (TPT) solution was incubated at 37 °C using a thermo-mixer. A 0.5 mL of dimethyl sulfoxide (DMSO) solution was mixed with 1.5 mL of the reaction solution at the regular intervals of 0, 12, 24, 36, 48, and 60 min to quench the OH radical generation. TPT captures OH radicals and generates a fluorescent compound, i.e., 2-hydroxyterephthalic acid (2-OHTA). The 2-OHTA fluorescence intensity was measured at an excitation/emission wavelength of 310/425 nm using a Fluorescence Spectrophotometer (Horiba Scientific; Edison, NJ, USA). The 2-OHTA concentration was determined by calibrating the instruments with a known concentration of a standard. The linear regression equation with (R² = 0.98) was used to estimate the rate of OH radical generation. OH radical generation in the DTT assay was measured separately, following [32 (link)].

This assay is based on a procedure described by[10 (link)]. Vesicles were prepared in 20 mM Na-MOPS, 0.8 mM calcein, 1 mM CoCl₂, 90 mM NaCl, pH 7.5. Unencapsulated material was removed on a Sephadex G-75 column, eluted with 20 mM Na-MOPS, 50 mM NaCl, 10 mM EDTA, pH 7.5. Leakage of calcein was followed for up to 24 hours on a Jobin Yvon fluorescence spectrophotometer with excitation at 495 nm and emission at 515 nm (slit widths of 3 nm for excitation and emission). To achieve 100% leakage (I_max), 0.25% (v/v) Triton X-100 was added. The percentage of calcein leakage was calculated by:
$\%Leakage=\frac{\left(I_t-I_0\right)}{(I_{max}-I_0)}x100\%$
where I_t is the intensity measured at the indicated time point and I₀ the intensity at the start of the experiment. To determine the release of calcein via AMPs, we tested melittin (dissolved in water) and alamethicin (dissolved in methanol); the final methanol concentration was kept below 1% (v/v), and controls showed that methanol had no influence on the leakage.

Fluorescence spectroscopy was applied to investigate the tertiary protein structure of IFX and potential deviations thereof due to compounding [57 (link)]. Filtered samples were further diluted 10× in PBS pH 6.8 (150 mM) containing 0.05% (w/v) polysorbate 20. This sample was transferred to a 10.00 mm fluorescence quartz cuvette. The sample in the secured quartz cuvette was stirred magnetically to prevent photobleaching. The intrinsic fluorescence of the sample was analyzed with a fluorescence spectrophotometer (Photon Technology International, Inc., Birmingham, AL, USA) using an excitation wavelength of λ = 295 nm, a slit width of 2.5 nm, sample temperature of 20.0 °C, and the emission spectrum at λ = 300–360 nm was analyzed. The average (n = 3) emission spectrum of ColoPulse-placebo was subtracted from the emission spectrum of the ColoPulse-IFX tablets (Table 2) to correct for the effects of the medium and tablet excipients during analysis.

To prepare monomeric actin, pyrene-labeled RMA and gel-filtered, unlabeled RMA were centrifuged in parallel for 1 h at 90,000 rpm in a TLA100 rotor (Beckman Coulter). The upper 75% of each supernatant was carefully removed, actin concentrations were redetermined, and labeled and unlabeled RMA were mixed at a 1:19 ratio. Assembly reactions were performed in a 60 μl final volume and contained final concentrations of 2 μM G-actin (5% pyrene labeled). The actin mixture was converted to Mg-ATP-actin 1 min before use, and then 42 μl of actin was mixed with 15 μl of proteins or control buffer, plus 3 μl of 20 initiation mix (40 mM MgCl2, 10 mM ATP, 1 M KCl) to initiate polymerization. Pyrene fluorescence was monitored at 365 nm excitation and 407 nm emission at 25° in a fluorescence spectrophotometer (Photon Technology International, Lawrenceville, NJ).