All MhuD samples for rR measurements were concentrated to ∼150 to 200 μM protein concentration in Raman buffer (100 mM potassium phosphate, pH 7.5). In total, 100 μl of each sample was transferred to a Wilmad 5 mm Economy NMR tube for rR measurements. The samples for ferric state studies at different pH were exchanged into the appropriate buffers: 100 mM potassium phosphate pH 5.5 or 100 mM borate pH 9.1. To prepare the ferrous and ferrous-CO samples, NMR tubes containing ferric samples in Raman buffer were septum-sealed to ensure an anaerobic environment. Samples were degassed under flow of argon for 25 to 30 min. For ferrous-CO samples, the NMR tubes were then filled with the appropriate isotopes of CO gas. The samples were then reduced by addition of approximately threefold molar excess of anaerobically prepared 50 mM sodium dithionite using a gas-tight syringe and rR spectra were measured immediately.
5 mm economy nmr tube
Manufactured by Wilmad
The 5 mm Economy NMR tube is a type of laboratory equipment used for nuclear magnetic resonance (NMR) spectroscopy. It is a glass tube with a diameter of 5 millimeters, designed to hold a sample for analysis in an NMR spectrometer.
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2 protocols using 5 mm economy nmr tube
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Resonance Raman Spectroscopy of MhuD
2
Resonance Raman Spectroscopy of Heme Oxygenase-1
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All hHO-1 samples for rR measurements were ~50-100 μM protein concentration in 100 mM potassium phosphate, pH 7.5. To prepare inhibitor-bound ferric samples, 20-fold excess of a given inhibitor was added to ferric enzyme. 100 μL of each sample was transferred to a Wilmad 5 mm Economy NMR tube for rR measurements. To prepare ferrous samples, NMR tubes containing ferric inhibitor-free or inhibitor-bound hHO-1 were closed by a septum. Each sample was degassed under flow of argon for 25-30 minutes to ensure an anaerobic environment. The samples were then reduced by addition of ~3-fold molar excess of anaerobically prepared sodium dithionite using a gas-tight syringe. To prepare the ferrous CO adducts, the reduced sample was then exposed to appropriate isotopes of CO gas. Resonance Raman spectra were measured immediately after formation of ferrous or ferrous-CO adducts. It is noted that the same ferrous CO spectra were obtained when inhibitor-bound ferric hHO-1 was first saturated with CO gas then reduced by addition of sodium dithionite solution.
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