Specord 200 spectrophotometer

In order to confirm our hypothesis, we designed three types of cryogels: a pullulan control sample and two pullulan–dopamine materials, as follows. First, the control sample was based solely on oxidized pullulan (PO) and was obtained by freeze-drying a dialdehyde pullulan solution containing 1 g of polysaccharide. Second, two oxidized pullulan–dopamine cryogels were prepared using two different mechanisms of dopamine incorporation, i.e., either in the pullulan solution or in the freeze-dried pullulan scaffold. Therefore, 0.2 g of dopamine was added to an oxidized pullulan solution and the mixture was kept in contact for 2 h in order to allow the Schiff base reaction. Next, the solution was frozen and lyophilized, thus obtaining the POD cryogel. By comparison, a POD1 cryogel sample was obtained by immersing a freeze-dried pullulan scaffold into a 0.2% dopamine solution in Millipore water. The mixture was kept in contact for 2 h, after which it was frozen and lyophilized. The amount of dopamine adsorbed by the POD1 cryogel was determined by means of UV-VIS spectroscopy (SPECORD 200 spectrophotometer, Analytik Jena, Jena, Germany).

The diastatic activity was determined by the Phadebas method in which the α-amylase activity is expressed as the diastase number, and is reported in Schade units [40 ]. One Schade unit corresponds to the enzyme activity contained in 1 g of honey, which can hydrolyze 0.01 g of starch in 1 hour at 40 °C. The procedure was as follows: first, 1 g of the analyzed honey was weighed, transferred to a 100 mL volumetric flask, and filled up to its volume with 0.1 M acetate buffer at pH = 5.2. Then, 5 milliliters of the sample was transferred to the test tube, placed in a water bath at 40 °C, and after 15 min a Phadebas tablet was added to the solution. The solution was mixed and heated again in a water bath for 30 min. After this time, 1 mL of 0.5 M sodium hydroxide solution was added to complete the enzymatic reaction. Next, the solution was filtered through a filter paper (φ = 70 mm) and the absorbance at 620 nm was measured using a Specord 200 spectrophotometer (Analytic Jena, Jena, Germany) as it is in proportion to the enzyme activity in the analyzed honey sample. The measurements were collected in triplicate and the final result is their average.

Proton release during the hydrolysis of dUTP into dUMP and PP_i was followed continuously at 559 nm at 20 °C using a Specord 200 spectrophotometer (Analytic Jena, Germany) and 10 mm path length thermostatted cuvettes as described previously^{21 (link),42 (link),48 (link)}. Reaction mixtures contained 50 nM hDUT enzyme and varying concentrations of Stl in activity buffer (1 mM HEPES (pH 7.5), 5 mM MgCl₂, 150 mM KCl, 40 μM Phenol Red indicator). The reaction started with the addition of 30 μM dUTP after 5 min pre-incubation of the two proteins in the assay buffer. Initial velocity was determined from the slope of the first 10% of the progress curve. Stl inhibition data were fitted to the quadratic binding equation describing 1:1 stoichiometry for the dissociation equilibrium with no cooperativity^{42 (link)}.

Lipid peroxidation was estimated by the thiobarbituric acid (TBA) method based on production of the MDA [81 (link)]. Briefly, 0.5 mL of supernatant and 1 mL of 0.5% TBA in 20% TCA were mixed and incubated at 95 °C. After 30 min of incubation, samples were cooled in an ice bath and centrifuged at 14,000× g for 15 min at 4 °C. Collected supernatants were used to measure the absorbance at 532 and 600 nm in a Specord 200 spectrophotometer (Analytic Jena, Jena, Germany) against the blank (0.5% TBA in 20% TCA). The accumulated MDA was calculated using an extinction coefficient of 155 mM⁻¹ cm⁻¹ and expressed as nmols per gram of FW.

The crude extract was subjected to a single step precipitation using 65 % (NH₄)₂SO₄ (Bio Xtra, >99 %; Sigma-Aldrich) and kept overnight at 4 °C. The pellet was recovered by centrifugation at 27,000 rpm for 15 min at 4 °C and dissolved in 10 ml of the same extraction buffer and termed as ammonium sulfate extract (ASE). Ten ml of ASE was dialyzed against the extraction buffer using dialyses membrane (Dialyses membrane-70, MWCO; 12–14 kD) procured from Hi-Media. Dialyses was performed twice against 1,000 ml extraction buffer, first at room temperature and again dialysed against 1,000 ml of extraction buffer at 4 °C overnight. The resultant extract was recovered from the dialyses membrane and filtered through 0.45 µm filter.
DEAE-Cellulose from Sisco Research Laboratory (SRL) was used for anion exchange chromatography. A column (30 × 2 cm) was prepared for purifying the phycocyanin, and equilibrated with 150 ml of acetate buffer (pH-5.10). Dialyzed filtered sample (10 ml) was placed on the column. A linear gradient of acetate buffer with pH ranging from 3.76 to 5.10 was used to developed the column and elutes were collected in 5 ml fractions. Flow rate was kept 20 ml h⁻¹. Absorption spectrum was also determined by scanning the sample in the range of 300–750 nm by using Specord 200 spectrophotometer (Analytikjena, Germany).

Twelve plants in each group were selected, four were pooled as a repeat, and three biological replicates were set. The plant heights of each group of Q9 and 65 plants were measured using a meter stick (Qinghai, China). Moreover, the chlorophyll contents of potato leaves were measured using a SPECORD 200 spectrophotometer (Analytik Jena, Germany) according to a previously reported method [22 (link), 23 ]. Leaf extractions containing chloroplast pigments were measured for specific light absorption at 665 and 649 nm. The chlorophyll content was calculated as follows: Chl = 13.95 × A_665–6.88 × A_649 + 24.96 × A_649–7.32 × A_665. The dry matter of the roots, shoots, and leaves was measured by using the following method: the divided tissues were heated in an oven (105 °C, 30 min) and then dried to constant weight (70 °C, 8 h); the dry matter was weighed on an electronic balance (METTLER TOLEDO, Shanghai, China). Statistical analysis of plant morphological data was conducted using SPSS 19.0 (IBM, Chicago, IL, USA). N accumulation was also determined using an ultraviolet spectrophotometer and a methylthymol colorimetric method. Statistical results were obtained by one-way analysis of variance (ANOVA) followed by Tukey’s test to evaluate significant treatment effects.

The investigated porphyrins were obtained according to the methods previously reported by us [27 (link),28 ,29 ]. Because of their structural configurations, these compounds have demonstrated an excellent solubility in biologically friendly media, and long-term stability in PEG 200, a non-toxic and green pharmacologically accepted solvent [43 (link)]. The stock solutions of porphyrins (10 mM) were prepared in PEG 200, and were further diluted for experiments to 10 μM in PEG 200, PEG 200/PBS (1/1000), or in cell culture medium, depending on the type of experiment. For preventing uncontrolled photodegradation, porphyrin stocks in PEG 200 were kept in “dark” conditions at room temperature. Working solutions of porphyrins and cells loaded with porphyrins were manipulated in “dark” conditions as well, except when PDT was performed.
The spectral behavior of the porphyrinic structures was assessed by UV-Visible and fluorescence spectroscopy. The UV–Vis spectra of the porphyrins were registered with a Specord 200 spectrophotometer (Analytik Jena, Jena, Germany). Fluorescence spectra were registered using a steady-state Jasco FP 6500 spectrofluorometer (JASCO Co., Ltd., Kyoto, Japan) in 10 mm path length quartz cuvettes.