Cysteine Hydrochloride → Nitrite
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N([S:3][CH2:4][C@@H:5]([C:7]([OH:9])=[O:8])[NH2:6])=O.Cl.N[C@H](C(O)=O)CS.[N:18]([O-:20])=[O:19].[Na+]>[N]=O.Cl.O>[NH2:6][C@H:5]([C:7]([OH:9])=[O:8])[CH2:4][SH:3].[N:18]([O-:20])=[O:19]N([S:3][CH2:4][C@@H:5]([C:7]([OH:9])=[O:8])[NH2:6])=O.Cl.N[C@H](C(O)=O)CS.[N:18]([O-:20])=[O:19].[Na+]>[N]=O.Cl.O>[NH2:6][C@H:5]([C:7]([OH:9])=[O:8])[CH2:4][SH:3].[N:18]([O-:20])=[O:19]Hb was reacted with 10-fold molar excess S-nitrosocysteine (CYSNO) which was synthesized with modification of standard procedure (see, for example, Stamler, J. S. and Feelisch, M., “Preparation and Detection of S-Nitrosothiols,” pp. 521-539 in Methods In Nitric Oxide Research (M. Feelisch and J. S. Stamler, eds.), John Wiley & Sons Ltd., 1996) as follows. L-cysteine hydrochloride (1.1 M) dissolved in 0.5 N HCl/0.5 mM EDTA was reacted with an equal volume of 1 M NaNO2 (sodium nitrite) dissolved in water, to form CYSNO (the ratio of cysteine to nitrite influences the SNO-Hb product and activity profile). The concentration of CYSNO was then adjusted by dilution in 200 mM phosphate buffer (PBS), pH 8.0, to yield a working CYSNO solution (pH 6-7). This was then reacted at room temperature with a 20-fold volume excess of Hb (in borate, pH 9.2), resulting in a final 10-fold molar excess of CYSNO over Hb (ratio influences product critically). Following the incubation (periods determined by the desired synthetic preparation; i.e., a desired ratio of SNO/tetramer; desired met- to oxy- to nitrosyl-Hb ratios; polynitrosated or non-polynitrosated; for example, 10 min. preferred time for SNO-oxyHb with 2 SNO per tetramer; see below), the reaction mixture was rapidly added to a column of fine Sephadex G-25 (20 to 30-fold volume excess over the reaction mixture) preequilibrated with 100 mM PBS pH 7.4, 0.5 mM EDTA. Typically, a 150 μl sample of the mixture was added to a 4.5 ml column measuring 12 mm (inner diameter). The column was then centrifuged at 1200 g for 60 seconds and the effluent collected in a 1.5 ml airtight plastic vial that was subsequently kept on ice and protected from light.
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