We diluted 8.8 M H2O2 stock solution, stored at −20°C, to 200 mM in deionized water just prior to the oxidation reaction. We incubated 100 μM Rucred or RucNterm-red at 37°C for 3 min, after which 50 μM copper chloride was added, followed by 2 mM H2O2. The reaction mixture was incubated for 10 min; H2O2 and copper chloride were removed using a Zeba Spin 7K MWCO desalting centrifuge column (Fisher Scientific) preequilibrated with the original Rucred storage buffer according to the manufacturer’s instructions. Treatment of Ruc with 2 mM diethylamine NONOate (DEANO, a nitric oxide donor) (Sigma-Aldrich) or NaOCl (Fig. S1B ) was performed identically except that Ruc was treated for 20 min (NaOCl) or 2 h (DEANO) as described for Hsp33 (62 (link)).
Diethylamine nonoate
Manufactured by Merck Group
Sourced in Belgium
Diethylamine NONOate is a chemical compound used in research laboratory settings. It functions as a nitric oxide (NO) donor, releasing controlled amounts of NO, which is a signaling molecule with various biological roles. The core function of Diethylamine NONOate is to provide a reliable source of NO in in vitro experiments and assays.
Automatically generated - may contain errors
Lab products found in correlation
Hydrogen peroxide, by Merck Group (2 mentions)
Sodium azide, by Thermo Fisher Scientific (1 mentions)
Potassium cyanide, by Thermo Fisher Scientific (1 mentions)
Diethylenetriaminepentaacetic acid, by Merck Group (1 mentions)
Ferrous ammonium sulfate, by Merck Group (1 mentions)
Amicon centrifugal filter, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Glycerol, by Thermo Fisher Scientific (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (1 mentions)
D glucose, by Thermo Fisher Scientific (1 mentions)
Ammonium acetate, by Thermo Fisher Scientific (1 mentions)
Amplex red, by Thermo Fisher Scientific (1 mentions)
Silver nitrate, by Merck Group (1 mentions)
Horseradish peroxidase, by Merck Group (1 mentions)
Malic acid, by Merck Group (1 mentions)
Potassium ferricyanide, by Merck Group (1 mentions)
Imidazole, by Merck Group (1 mentions)
Chloramphenicol, by Merck Group (1 mentions)
Xanthine oxidase, by Merck Group (1 mentions)
5 protocols using diethylamine nonoate
1
Oxidation of Ruc Proteins by H2O2 and Oxidants
2
Synthesis and Handling of Hazardous Compounds
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Hydrogen peroxide and diethylamine NONOate were purchased from Sigma. Potassium cyanide and sodium azide were purchased from Fisher-Scientific.
3
Comprehensive Biochemical Reagent Inventory
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Ammonium acetate, acetonitrile, phenylalanine, glycerol, and d -glucose were purchased from Fisher Scientific. Arabinose, 5-aminolevulinic acid (5-ALA), ascorbic acid, biotin, bovine liver catalase, chloramphenicol, citraconic acid, copper (II) sulfate, 5′deoxyadenosine (5 dA), 5′-deoxy-5’-(methylthio)adenosine (MTA), diethylamine NONOate, diethylenetriamine pentaacetic acid (DTPA), dithiothreitol (DTT), ferrous ammonium sulfate (FAS), horseradish peroxidase, hydrogen peroxide, imidazole, isopropyl β-d -1-thiogalactopyranoside (IPTG), lipoic acid, malic acid, potassium ferricyanide, S-adenosylmethionine (SAM), silver nitrate, superoxide dismutase (SOD), sodium dithionite, xanthine, xanthine oxidase, histidine, cystine, isoleucine, methionine, tyrosine, tryptophan, and valine were obtained from Sigma. Amicon centrifugal filters were from Millipore, and Amplex Red was from Invitrogen. His GraviTrap™ was purchased from GE Healthcare.
4
Mouse Lung Prostacyclin Release Bioassay
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A human platelet bioassay was used to measure bioactive prostacyclin levels released from segments of mouse lung parenchyma (≈10 mm3). Platelet-rich plasma (PRP) and platelet-poor plasma were separated from human blood by centrifugation (PRP: 230g, 15 minutes; platelet-poor plasma: 8000g, 2 minutes). PRP was preincubated with aspirin (30 μM, 30 minutes prior; Sigma) and diethylamine NONOate (10 μM, 1 minute prior; Sigma) to sensitize platelets to prostacyclin. Lung segments were added to individual wells of 96-well microtiter plates containing PRP and preincubated for 1 minute, before stimulation of platelets and tissues with A23187 (30 μM) and vigorous mixing (1200 revolutions per minute, BioshakeIQ; Q Instruments, Germany). After 5 minutes, the tissue segments were removed and the absorbance of each well at 595 nm measured by spectrophotometer and the amount of platelet aggregation calculated by reference to the absorbance of unstimulated PRP (0% aggregation) and platelet-poor plasma (100% aggregation).
5
Vasodilation and Anesthetic Protocols
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Sodium pentobarbital (Nembutal®) was obtained from Sanofi (Brussels, Belgium), indomethacin from Federa (Belgium), L-NAME, phenylephrine hydrochloride (PE) and diethylamine NONOate (DEANO) from Sigma-Aldrich (Bornem, Belgium), diltiazem hydrochloride from TOCRIS (Bristol, United Kingdom).
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