Decomposition of hydroxylamine by hemoglobin

Abstract

The reaction between hydroxylamine (NH 2OH) and human hemoglobin (Hb) at pH 6–8 and the reaction between NH 2OH and methemoglobin (Hb +) chiefly at pH 7 were studied under anaerobic conditions at 25 °C. In presence of cyanide, which was used to trap Hb +, Hb was oxidized by NH 2OH to methemoglobin cyanide with production of about 0.5 mol NH + 4/mol of heme oxidized at pH 7. The conversion of Hb to Hb + was first order in [Hb](or nearly so) but the pseudo-first-order rate constant was not strictly proportional to [NH 2OH]. Thus, the apparent second-order rate constant at pH 7 decreased from about 30 m −1 × s −1 to a limiting value of 11.3 m −1 × s −1 with increasing [NH 2OH]. The rate of Hb oxidation was not much affected by cyanide, whereas there was no reaction between NH 2OH and carbonmonoxyhemoglobin (HbCO). The pseudofirst-order rate constant for Hb oxidation at 500 μ m NH 2OH increased from about 0.008 s −1 at pH 6 to 0.02 s −1 at pH 8. The oxidation of Hb by NH 2OH terminated prematurely at 75–90% completion at pH 7 and at 30–35% completion at pH 8. Data on the premature termination of reaction fit the titration curve for a group with p K = 7.5–7.7. NH 2OH was decomposed by Hb + to N 2, NH + 4, and a small amount of N 2O in what appears to be a dismutation reaction. Nitrite and hydrazine were not detected, and N 2 and NH + 4 were produced in nearly equimolar amounts. The dismutation reaction was first order in [Hb +] and [NH 2OH] only at low concentrations of reactants and was cleanly inhibited by cyanide. The spectrum of Hb + remained unchanged during the reaction, except for the gradual formation of some choleglobin-like (green) pigment, whereas in the presence of CO, HbCO was formed. Kinetics are consistent with the view advanced previously by J. S. Colter and J. H. Quastel ((1950) Arch. Biochem. 27, 368–389) that the decomposition of NH 2OH proceeds by a mechanism involving a Hb/Hb + cycle (reactions [1] and [2]) in which Hb is oxidized to Hb + by NH 2OH.