MetHb-nitrite was prepared in 0.01M PBS buffer by adding 300 μM, 1.5 mM, 3mM, or 6mM nitrite to 300 μM methemoglobin. Dissociation constants (Kd) were calculated by fitting simulations of the high spin MetHb, the low spin MetHb, and the two low spin MetHb-nitrite species to the experimental data and calculating concentrations of each species from the simulations. Inset shows Kd values for samples prepared at pH 6.5 on a smaller scale.