To evaluate the NOS activity, oxyhemoglobin assay was performed. iNOS activity was assessed in calcium-free conditions using an assay mixture contained l -arginine (10 μM), NADPH (100 μM), tetrahydrobiopterin (6.5 μM), and oxyhemoglobin (3 mM) in HEPES (100 mM, pH 7.5). The assays were initiated with lysate addition (40 μg of protein) in a final volume of 200 µL. Nitric oxide reacts with oxyhemoglobin to yield methemoglobin which is detected at 576 nm (e = 12,000 M−1cm−1) on a Perkin-Elmer LamdaBIO UV–vis spectrophotometer.
Lamdabio uv vis spectrophotometer
Manufactured by PerkinElmer
Sourced in Italy
The LamdaBIO UV–vis spectrophotometer is a laboratory instrument used to measure the absorption or transmission of light in a sample across the ultraviolet and visible light spectrum. It provides quantitative analysis of various chemical and biological samples.
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4 protocols using lamdabio uv vis spectrophotometer
1
Quantifying iNOS Activity via Oxyhemoglobin Assay
2
Quantifying Nitric Oxide Production by NOS
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Nitric oxide formation from NOS was monitored by the oxyhemoglobin assay as described previously 25 (link). A typical assay mixture for NOS contained 10 μM l -arginine, 1 mM MgCl2, 100 μM NADPH, 6.5 μM tetrahydrobiopterin, and 3 mM oxyhemoglobin in 100 mM HEPES (pH 7.5) iNOS activity was assessed as described above, but in calcium-free conditions. All assays were in a final volume of 1 ml and were initiated with enzyme. Nitric oxide reacts with oxyhemoglobin to yield methemoglobin which is detected at 576 nm (e = 12,000 M−1 cm−1) on a Perkin-Elmer LamdaBIO UV–vis spectrophotometer.
3
Intracellular NOS Activity Assay
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Oxyhemoglobin assay was used to monitor the intracellular NOS enzyme activity [27 (link)]. The assay was initiated with enzyme in a total volume of 1 mL. NO reacts with oxyhemoglobin to yield methemoglobin that is detected at 576 nm (e = 12,000 M−1 cm−1) using a Perkin-Elmer LamdaBIO UV–Vis spectrophotometer.
4
Nitric Oxide Synthesis Assay Using Oxyhemoglobin
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The oxyhemoglobin assay was performed to detect nitric oxide formation from NOS as described above [47 (link)]. The reaction mixture for the evaluation of NOS activity was constituted by CaCl2 (1.6 mM), l -arginine (10 µM), calmodulin (11.6 mg/mL), tetrahydrobiopterin (6.5 µM), dihydronicotinamide-adenine dinucleotide phosphate (NADPH, 100 µM) and oxyhemoglobin (3 mM) in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 100 mM) (pH 7.5) in a final volume of 1 mL. iNOS activity was performed in calcium-free conditions. The quantization of methemoglobin as the product of the reaction between nitric oxide and oxyhemoglobin was detected at 576 nm (e = 12.000 M−1·cm−1) using a Perkin-Elmer LamdaBIO UV-Vis spectrophotometer (Perkin-Elmer, Milano, Italy).
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