We measured total phenolic content (TPC) with a modified version of the Folin-Ciocalteu method [21 (link)] using 0–0.1 mg/mL catechin as a standard. Briefly, 100 μL of sample or standard was combined with 100 μL of Folin-Ciocalteu reagent and 100 μL of 2% Na2CO3 solution. We allowed the mixture to sit for 60 min before reading absorbance at 750 nm using an Ultrospec Visible Plate Reader II 96 (GE Healthcare Ltd., England) and calculating the concentration of plant sprouts' aqueous extracts (PSAE) as catechin equivalents per gram of plant sprouts.
Ultrospec visible plate reader 2 96
Manufactured by GE Healthcare
Sourced in United Kingdom
The Ultrospec Visible Plate Reader II 96 is a laboratory instrument designed for absorbance measurements in 96-well microplates. It is capable of measuring the optical density of samples within the visible light spectrum.
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5 protocols using ultrospec visible plate reader 2 96
1
Measuring Total Phenolic Content in Plant Sprouts
2
Enzymatic Hydrolysis Potential of BaLPMO10A
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To examine the hydrolysis potential of the purified enzyme against PNPC, a reaction was carried out in 50 mM sodium acetate buffer at a pH of 6.0, 5 µM BaLPMO10A, 1 mM PNPC. The reaction mixture was incubated at 50 °C over periods of 0, 6, 12, 24, 36, and 48 h. The reaction was terminated by adding 1M Na2CO3. The amounts of products formed during the reaction were determined by measuring the absorbance at 405 nm using a microplate reader (Ultrospec visible plate reader II 96, GE Healthcare BioScience). Finally, a p-nitrophenol standard curve was used to quantify the final concentration of the products released from the reaction [48 (link)]. Moreover, the degradation potential of the purified enzyme against CMC was examined. The reaction mixture consisted of 0.5% w/v CMC and 5 µM BaLPMO10A, and was incubated in a 1.5 mL Eppendorf tube with a total reaction volume of 250 µL in 50 mM sodium acetate buffer with a pH of 6.0. The reaction was carried out at 50 °C over periods of 0, 6, 12, 24, 36, and 48 h. The reaction was terminated by passing the reaction mixture through a filter with a pore size of 0.22 µm. The final concentration of the products formed was sing buy the PAHBAH assay method [32 (link)].
3
Polyamine Inhibition of LSD1, LSD2 and MAO
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LSD1 inhibition assay was performed using the peroxidase-coupled reaction method essentially as described previously.30,31 (link) Human LSD1 (2.8 μM) was incubated with serial dilutions of each polyamine in 50 mM HEPES-Na (pH 7.5) buffer containing 400 μM 4-aminoantipyrine, modified Trinder's reagent TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, sodium salt, dihydrate), and 40 μg mL−1 horseradish peroxidase at 25 °C for 10 min. The reaction mixture was subsequently incubated with 83 μM H3(1–20)K4-dimethylated (K4me2) peptide for 30 min. Inhibition assays for LSD2, MAO-A and MAO-B were performed in a similar manner to the LSD1 inhibition assay, using 167 μM H3(1–20)K4me2 peptide as the substrate for 2.7 μM LSD2, and 50 and 150 μM tyramine as the substrate for 1.4 and 2.8 μM MAO-A and MAO-B, respectively. Absorbance at 562 nm of the hydrogen peroxide by-products generated by H3(1–20)K4me2 demethylation or tyramine oxidation was measured with a 96-well microplate reader (Ultrospec Visible Plate Reader II 96; GE Healthcare). Steady-state reaction ranges from 0 to 10 min (LSD1, LSD2 and MAO-A) or 0 to 30 min (MAO-B) were used for fitting analysis with the equation of competitive inhibition via steady-state kinetic analysis to obtain Ki with GraphPad Prism 6 software (version 6.0e).40 (link)
4
Enzymatic Assay for LSD1 Inhibition
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IC50 values of LSD1 demethylase inhibition were obtained by the peroxidase-coupled reaction method as described previously [35 (link), 36 (link)]. Briefly, 1 μM human LSD1 was incubated with serial dilutions of inhibitors in 50 mM HEPES-Na (pH 7.5) buffer containing 400 μM 4-aminoantipyrine, modified Trinder’s reagent TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, sodium salt dihydrate), and 40 μg/ml horseradish peroxidase at 25°C for 10 min. The reaction mixture was subsequently incubated with 100 μM K4-dimethylated H3 tail peptide (1–20) for 30 min. Absorption of the peroxidase by-product generated by lysine demethylation was measured at 562 nm with a 96-well microplate reader (Ultrospec Visible Plate Reader II 96; GE Healthcare). IC50 values were calculated with Prism 6 software (version 6.0e), using dose-response results in triplicate. LSD2 inhibition assays were performed using 200 μM K4-dimethylated H3 tail peptide (1–20) and 1 μM human LSD2. MAO inhibition assays were performed using 50 or 150 μM tyramine, 100 μg/ml MAO-A (Sigma-Aldrich) and 200 μg/ml MAO-B (Sigma-Aldrich).
5
Quantification of Total Phenolic Content
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We measured total phenolic content (TPC) with a modified version of the Folin–Ciocalteu method[13 (link)] using 0–30 mg/L chlorogenic acid as a standard. The determination of TPC was done with the colorimetric method as previously described.[13 (link)] Briefly, 100: L of sample or standard was combined with 100: L of Folin-Ciocalteu reagent and 100: L of 2% Na2CO3 solution. We allowed the mixture to sit for 60 min before reading absorbance at 750 nm using an Ultrospec Visible Plate Reader II 96 (GE Healthcare Ltd., England) and calculating the concentration of PSAE as chlorogenic acid equivalents per gram of dried plant seed.
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