Uv 2401pc uv vis recording spectrophotometer

UV-2401PC UV-Vis recording spectrophotometer (Shimadzu Corporation, Tokyo, Japan) was applied to obtain the spectra of purified polysaccharide. Each spectrum was at the average of five parallel scanning from 200 to 400 nm. The scan interval was 0.2 nm and the width of entrance slit was 12 nm.

For light-induced adduct formation accompanied by absorbance changes in the spectral region 425–520 nm, we used a photographic flash (Canon) as a light source. Adduct decay kinetics were measured by following absorbance at 447 nm in quartz cuvettes with 1 cm pathlength. A UV-2401PC UV-Vis recording spectrophotometer (Shimadzu) was used. In all measurements, the protein concentration was 25 µM.

A total of 1 × 10⁶ cells were resuspended in 200 µL of mannitol buffer (10 mM Tris-HCl pH 7.2; 225 mM mannitol; 75 mM sucrose; 0.1 mM EDTA) and homogenized by sonication for 5 s at 60% intensity with Microson Ultrasonic Cell Disruptor XL2000 (Misonix). Then, the homogenates were centrifuged at 650 × g for 20 min at 4 °C, and the protein concentration of the resulting supernatants was quantified by Bradford assay. Homogenates were brought to 1 mg/ml in mannitol buffer. NADH oxidation was determined after electron transfer to ubiquinone by monitoring the decrease in 340 nm absorbance^{64 (link),65 (link)}. Briefly, 20 µg of protein homogenate were mixed with 100 µM decilubiquinone, 3.75 mg/mL BSA, and 50 mM K₂HPO₄ pH 7.5 in the presence or absence of 12.5 µM of rotenone. The reaction was initiated by adding 100 µM NADH at 37 °C, and light absorbance was monitored at 340 nm for 3 min in a UV-2401PC UV-VIS recording spectrophotometer (Shimadzu Corporation).

UV–vis measurements used to determine rhodamine loading were carried out at 1 mg/mL in PBS using a UV-2401PC UV–vis recording spectrophotometer from Shimadzu. The rhodamine B content was calculated using a calibration curve for rhodamine B in PBS.

The NADH/NAD concentrations were analyzed using adapted methods of Wimpenny and Firth [26 (link)]. Bacterial cells were grown to ~1.0 OD₅₅₀ in LB broth (50 mL) containing 2 % glucose at 37 °C under aerobic and oxygen limiting conditions. These cells were pelleted, and either treated with 300 μL HCl (200 mM, pH 1.5) for NAD extraction or with 300 μL KOH (200 mM, pH 11.5) for NADH extraction. The cells were then incubated at 50 °C for 10 min, cooled to 4 °C, and then neutralized by adding 300 μL either 100 mM NaOH (for NAD) or 100 mM HCl (for NADH). After centrifugation, the supernatants were used for the NADH/NAD assays which were performed in a 1.5 ml cuvette as follows: 400 μL mixed solution (maintained at 30 °C) containing equal amount of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (4.2 mM), EDTA (40 mM), Tris (1 M, pH 8.0), and 95 % ethanol; 300 μL H₂O; 200 μL phenazine ethosulfate (33.2 mM); and 50 μL sample supernatant. The reaction was initiated by adding 50 μL yeast alcohol dehydrogenase II (500 U/mL), and the absorbance was read at 570 nm for 5 min using a UV-2401PC UV–VIS Recording Spectrophotometer (Shimadzu). All components except ethanol were used as the blank. All assays were performed in triplicate.