Reactions of deoxy-, oxy-, and methemoglobin with nitrogen monoxide. Mechanistic studies of the S-nitrosothiol formation under different mixing conditions

The reaction between hemoglobin (Hb) and NO* has been investigated thoroughly in recent years, but its mechanism is still a matter of substantial controversy. We have carried out a systematic study of the influence of the following factors on the yield of S-nitrosohemoglobin (SNO-Hb) generated from the reaction of NO* with oxy-, deoxy-, and metHb: 1) the volumetric ratio of the protein and the NO* solutions; 2) the rate of addition of the NO* solution to the protein solution; 3) the amount of NO* added; and 4) the concentration of the phosphate buffer. Our results suggest that the highest SNO-Hb yields are mostly obtained by very slow addition of substoichiometric amounts of NO* from a diluted solution. Possible pathways of SNO-Hb formation from the reaction of NO* with oxy-, deoxy-, and metHb are described. Our data strongly suggest that, because of mixing artifacts, care should be taken to use results from in vitro experiments to draw conclusion on the mechanism of the reaction in vivo.