反应详情

Hb was reacted with 10-fold molar excess S-nitrosocysteine (CysNO) which was synthesized immediately before use in high concentration by a 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), and is neutralized prior to addition to the hemoglobin solution by dilution in 100–200 mM PBS (pH 7.4 to 8.0, with 0.5 mM EDTA). The concentration of CysNO was then adjusted by dilution in PBS, pH 8.0, to yield a working CysNO solution (pH 6–7). Oxyhemoglobin (>100 μM in borate, pH 9.1–9.2) was S-nitrosylated by incubation with a 10-fold molar excess of CysNO over Hb (ratio influences product critically). Periods of incubation are 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. is a preferred time for SNO-oxyHb with 2 SNO per tetramer. The reaction was stopped by rapid transfer of the reaction mixture to a column of fine Sephadex G-25 (bed volume should be 10 to 30-fold excess over that of 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 800–1200 g for 60 seconds and the effluent containing purified SNO-oxyHb collected in a 1.5 ml airtight plastic vial that was subsequently kept on ice and protected from light.

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