Lambda bio 20 spectrophotometer

Difference spectroscopy (heme titrations) studies were done as described earlier [16] (link). Spectra were recorded on a Perkin-Elmer Lambda Bio20 spectrophotometer between 250–700 nm at a speed of 120 nm/min and a slit width of 1 nm. Heme was titrated in two separate experiments, for binding to BNT1 and BNTM. A hemin solution (2 mM in DMSO) was simultaneously titrated into (i) a solution of 5 µM of BNT1/BNTM in 3 mL of 200 mM HEPES, pH 7 and (ii) a reference cuvette containing HEPES buffer only. Heme was titrated in 2 µM increments. After each addition of heme, the samples in the two cuvettes were mixed and allowed to stand for 5 minutes to allow complete binding before recording of difference spectra. Difference absorption spectra were recorded after each incremental addition of heme. Concentration of DMSO was kept below 2% in all reaction mixtures. The difference spectra had their maxima and minima at 415 nm and 360 nm respectively. Heme-binding curves of BNT1 and BNTM were generated from the difference absorption spectra by plotting A₄₁₅ vs the moles of heme/mole of peptide template. Horizontal dotted lines were drawn to connect the plateau points of each graph to the Y axis. At the point of intersection, vertical arrows were drawn to obtain the values of heme/peptide.

The radical-scavenging activity was measured by the 2,2′-azino-nis (3-ethylbenzthiazoline-6-sulfuric acid (ABTS) radical cation discoloration assay [27 (link)]. The ABTS radical stock solution was prepared by dissolving the ABTS in water to a 7 mM concentration and by reacting this solution with 2.45 mM of potassium persulfate; the mixture was then left in the dark at room temperature for 12–16 h before use. The ABTS+• stock solution was diluted in water to an Abs of 0.70 ± 0.02 for the analysis and the reaction was started by the addition of 30 µL of CSS extract to 2.97 mL of ABTS+• radical solution.
The bleaching rate of ABTS+• in the presence of the sample was monitored at 25 °C at 734 nm using a Perkin Elmer (Boston, MA, USA) Lambda Bio 20 spectrophotometer, and the discoloration after 5 min was used for the calculation of the inhibition percentage.
Radical-scavenging activity was expressed as mol Trolox equivalents antioxidant capacity (TEAC-µmol of Trolox equivalents per g of sample) and calculated by the ratio of the correlation coefficient of the dose–response curve of the sample and the correlation coefficient of the dose–response curve of Trolox, the standard compound.

For pure DA, AlgOX and AlgOX-DA, UV-vis spectra were acquired with a PerkinElmer Lambda Bio 20 spectrophotometer at a wavelength range of 230–340 nm. Prior to acquiring the spectra, pure DA aqueous solution was prepared at 0.05 mg/mL at 25 °C, and AlgOX and AlgOX-DA were dissolved, obtaining the final concentrations of 1 mg/mL and 0.25 mg/mL, respectively. A slight sonication was required for complete dissolution for AlgOX-DA.

The scavenging activity for DPPH (Sigma, Milan, Italy) free radicals was measured according to Brand–Williams (1995), with some modifications. To 2.9 mL of a 10 × 10⁻⁵-M solution of DPPH in MeOH, 0.1 mL of a properly diluted MeOH sample test solution was added and vortexed, that is, different dilutions of each test sample (019, 014) were prepared to determine the actual IC50 values. After 30 min of incubation in the dark at room temperature, the UV absorbance (Perkin-Elmer Lambda Bio 20 Spectrophotometer) of each sample was recorded at 515 nm against the blank (reagent solution without the test sample). All determinations were carried out in triplicate. The inhibition percentage (I%) of DPPH radicals in the test samples was calculated with the following equation: I% = [(A_blank − A_sample)/A_blank] x 100, where A_blank is the absorbance of the blank solution at t = 0, and A_sample is the absorbance of the test sample at t = 30 min. The IC50 concentration is the amount of drug necessary to give 50% inhibition of the DPPH radicals. Acteoside, quercetin, and harpagoside were used as reference compounds.

The radical scavenging activity was measured by the ABTS [2,2′-azino-nis (3-ethylbenzthiazoline-6-sulfuric acid)] radical cation decoloration assay, as described by Re et al. (14 (link)). The bleaching rate of ABTS^+• in the presence of the sample was monitored at 734 nm using a Perkin Elmer (Boston, MA, USA) Lambda Bio 20 spectrophotometer. The ABTS^+• stock solution was diluted either in water or ethanol up to an Abs of 0.70 ± 0.02 for the analysis of the aqueous and apolar extracts, respectively. Volumes of 2.97 and 2.88 μL of ABTS^+• solution were used for hydrophilic and lipophilic extracts, respectively. The reaction was started by the addition of 30 and 120 μL of hydrophilic and lipophilic extracts to aqueous and ethanolic ABTS solution, respectively. The ABTS^+• bleaching was monitored at 30°C and the decoloration after 5 min was used as the measure of antioxidant activity. Radical scavenging activity was measured as Trolox Equivalents Antioxidant Capacity (mmol of Trolox eq. per 100 g of sample) and calculated by the ratio of the correlation coefficient of the dose–response curve of the sample and the correlation coefficient of the dose–response curve of the standard compound. Trolox was used as standard for the calculation of the radical scavenging activity for both the water soluble (TEAC_aq) and liposoluble extracts (TEAC_lipo).