Cary 300 spectrophotometer

Absorption and fluorescence spectra of the compounds were recorded in air-saturated solutions at ambient temperature with a Cary 300 spectrophotometer (Agilent Technologies, Victoria, Australia) and Eclipse Cary spectrofluorimeter (Agilent Technologies, Victoria, Australia). All measured fluorescence spectra were corrected for the nonuniformity of detector spectral sensitivity. Coumarin 481 in acetonitrile (fluorescence quantum yield φ^fl = 0.08) [28 (link)] was used as a reference for the fluorescence quantum yield measurements.

All γPNA oligomers reported here were purchased from PNA Innovations Inc and gave satisfactory HPLC and MS data. This vendor is no longer in business. Concentrations of struc_γPNA (ε₂₆₀ = 146,700 M⁻¹ cm⁻¹) and unstruc_γPNA (ε₂₆₀ = 94,400 M⁻¹ cm⁻¹) were determined by UV-vis absorption on a Cary 300 spectrophotometer (Agilent, Santa Clara, CA, USA). Sequences of all biotinylated target DNA oligonucleotides used in SPR direct binding experiments are also given below. DNA oligonucleotides were ordered from Integrated DNA Technologies (idtdna.com, Coralville, IA, USA).

Activities on PNP glycosides were analysed by a stopped assay as previously described (Larsbrink et al., 2011 (link)), using enzyme concentrations in the μM range for initial screens and several hours incubation. For initial rate kinetics on Gal-β-PNP for CjBgl35A, 42 nM enzyme was used and reactions were stopped after 10 min. Assays utilizing l-Fuc-α-CNP were monitored continuously for the release of 2-chloro-4-nitrophenolate using a Cary 300 spectrophotometer (Agilent Technologies). For the determination of the kinetic parameters of CjAfc95A, 22 nM enzyme was used. An extinction coefficient of 12 936 M⁻¹ cm⁻¹, determined from a standard curve, was used to calculate product concentration from A₄₀₅ values.

Growth estimation was based on the chlorophyll content measurements. For chlorophyll assay determination, an aliquot of the cell suspension was sampled and harvested by centrifugation for 5 min at 3000 g. Total Chl was extracted by heating the cell pellet with 2 mL of dimethyl sulfoxide (DMSO) for 10 min at 70 °C. From the cells attached to the carrier, chlorophyll was extracted by adding 2 mL of DMSO to the carrier with the cells and heating for 10 min at 70 °C. Concentration of chlorophyll was determined in the DMSO extracts with an Agilent Cary 300 spectrophotometer (Walnut Creek, CA, USA) using equations reported in [29 (link)].
The residual phosphate and nitrate contents were checked using Thermo Dionex ICS 1600 HPLC (Sunnyvale, CA, USA) with a conductivity detector and IonPac AS12A anionic analytical column (5 μm; 2 × 50 mm). The column temperature was maintained at 30 °C. The ions were eluted isocratically with 2.7 mM sodium carbonate/0.3 mM sodium bicarbonate buffer (flow rate of 0.3 mL/min).

Samples were reduced in an anaerobic glove bag (Coy) with 5 mM Na₂S₂O₄ for 15 min, and then desalted with a BioSpin6 column equilibrated with Buffer E (50 mM HEPES, pH 7.5, 150 mM NaCl, 5% (v/v) glycerol, 0.22 µm filtered). CO-saturated buffer (950 μM CO) was prepared by sparging 3 mL of anaerobic Buffer E for 15 min in a Reacti-Vial (Thermo Fisher Scientific). CO was added to the sample to achieve a final concentration of 425 μM. Nitric oxide was added to a concentration of 500 μM by addition of DEA NONOate, based on 1.5 moles of NO released per mole of NONOate. Protein-ligand complexes were incubated for 15 min at room temperature to establish equilibrium, and no further spectral changes were observed after this time. Samples were placed in a septum-sealed 1 cm pathlength quartz cuvette inside the glove bag, and UV–Vis spectra were recorded on a Cary 300 spectrophotometer (Agilent Technologies).