Half-life of nitric oxide in aqueous solutions with and without haemoglobin

Nitric oxide (NO) has been linked to many regulatory functions in mammalian cells. Studies of NO release are hampered by the short half-life of the molecule. In the blood, NO disappears within seconds because it binds avidly with haemoglobin (Hb). The relationship between Hb concentration and NO disappearance, however, has not been described. In this study we utilized an amperometric NO sensor (WPI, Sarasota, FL) to monitor continuously the disappearance of NO from an aqueous solution when Hb (free or as red blood cells) was added. The calibration and linearity of the NO sensor was checked frequently using a chemical reaction to generate a known concentration of NO. An aliquot of NO solution (prepared from authentic gas) was added to a glass beaker containing 20 ml saline to generate NO concentration of approximately 1200 nM. Under our experimental conditions (PO2 = 40 mmHg), NO concentration fell slowly over 20 min with a half-life of 445 s. However, when haemoglobin was added, NO disappeared rapidly in proportion to Hb concentration. The results suggest that rapid binding of NO to Hb occurs in a 4:1 ratio. The maximum rate constant of NO disappearance due to binding with Hb was 2 x 10(5) M-1 s-1. The 4:1 binding ratio between NO:Hb may be used as a tool to quantitate NO release in some biological assays. The study supports the notion that NO acts as an autocoid because it disappears rapidly in the presence of Hb and is not likely to act as a circulating humoral substance. The NO sensor was useful for monitoring of NO concentration in Hb free solutions, but its response time limits its use in blood.