Nitric oxide (NO) 1 is one of the 10 smallest, stable molecules of the hundreds of millions in nature (1). According to Stokes' Law, the diffusibility of a molecule in the condensed phase is inversely proportional to its molecular radius, which thus makes NO one of the most rapidly diffusible molecules known. Its diffusion constant (D) is approximately 3300 -3800 m 2 /s, whether measured in aqueous solution (2) or in intact tissue (e.g. brain (3)). Membranes and other hydrophobic structures in tissue are no barrier to diffusion of NO because of its solubility in hydrophobic phases (4).The reaction of free NO with oxyhemoglobin is rapid (bimolecular rate constant k ϭ 3.4 ϫ 10 7 M Ϫ1 s Ϫ1 (5)), and from this rate constant it can be calculated that the half-life of NO in the presence of a concentration of hemoglobin equivalent to that in the bloodstream (15 g/dl) would be very short, approximately 2 ϫ 10 Ϫ6 s. As we have pointed out previously (6, 7), the extremely rapid diffusibility of NO coupled with its rapid reaction with oxyhemoglobin apparently poses a difficulty in the postulate that free NO is the endothelium-derived relaxing factor.Using an electrochemical method, we describe here the results of measurements of the disappearance of NO upon reaction with either oxyhemoglobin in solution or oxyhemoglobin when contained within intact erythrocytes. We find that, as reported in 1927 for the reaction of O 2 with deoxyhemoglobin (8), the NO reaction with intact RBCs is considerably slower than with an equivalent concentration of free oxyhemoglobin. We present a mathematical analysis of this phenomenon, which demonstrates that the rate of the reaction of NO with intraerythrocytic hemoglobin is limited by the rate of diffusion of NO into the cell. From our data, we estimate that in whole blood the half-life of NO will be less than 2 ms, which, although quite rapid, is considerably longer than in the presence of free hemoglobin. EXPERIMENTAL PROCEDURESPreparation of NO Solution-6 ml of phosphate-buffered saline (PBS: 15 mM phosphate (potassium) plus 0.09% NaCl pH 7.4) in a plastic vial was used in preparing saturated NO solution. The solution was bubbled with argon gas (Aldrich) for 30 min and then changed to NO gas (Aldrich) for 20 min. The NO gas was passed first through a gaswashing bottle containing 1 M deaerated KOH solution.RBC and Free Hemoglobin Preparation-Blood was withdrawn from rats and centrifuged at 2300 ϫ g for 10 min. The plasma and buffy coat were discarded, and the RBC pellet was washed 3 times with PBS (pH 7.4). The packed RBCs then were added to PBS and the solution was stirred gently. Cells were counted with a hemocytometer and were stored on ice for use. To prepare free oxyHb, 2 ml of counted RBCs was centrifuged at 2300 g for 10 min (4°C). The packed RBCs were then added to 40 ml of 5 mM phosphate solution (pH 8), stirred and allowed to incubate for 30 min for hemolysis.Electrochemical Measurements-All electrochemical measurements were carried out at 25 Ϯ 2°C by a BAS 100B electrochemical a...

We demonstrate herein dramatic acceleration of aqueous nitric oxide (NO) reaction with O 2 within the hydrophobic region of either phospholipid or biological membranes or detergent micelles and demonstrate that the presence of a distinct hydrophobic phase is required. Per unit volume, at low amounts of hydrophobic phase, the reaction of NO with O 2 within the membranes is approximately 300 times more rapid than in the surrounding aqueous medium. In tissue, even though the membrane represents only 3% of the total volume, we calculate that 90% of NO reaction with O 2 will occur there. We conclude that biological membranes and other tissue hydrophobic compartments are important sites for disappearance of NO and for formation of NO-derived reactive species and that attenuation of these potentially damaging reactions is an important protective action of lipid-soluble antioxidants such as vitamin E.Nitric oxide (NO) is an important mediator and messenger in mammalian systems and subserves an astonishing variety of roles in physiology and pathophysiology (1). One of its distinctive properties is its relatively short half-life (reported to be on the order of several seconds) in biological systems, which determines its spatial range and temporal extent of actions (2). One generally recognized mechanism for the disappearance of NO is reaction with O 2 , which is responsible for the formation of nitrite as a product of NO oxidation. Intermediates in this reaction are responsible for nitrosative reactions that result in the formation of biologically important species such as nitrosamines and nitrosothiols (2).The aqueous reaction of NO with dioxygen occurs with the following overall stoichiometry:and the rate of disappearance of NO is given bywith k ϭ 2 ϫ 10 6 M Ϫ2 ⅐s Ϫ1 at 25°C (3, 4). Because NO is approximately nine times more soluble in a hydrophobic solvent such as hexane than in water (5, 6), we [and others (7, 8)] have suspected that the presence of a hydrophobic phase (such as the interior of a lipid bilayer membrane) might accelerate the autooxidation of NO because of the concentration of reactants within the hydrophobic phase. Thus, biological membranes may act as a ''lens'' that can focus and magnify the autooxidation of NO. That is, even if the intrinsic rate constant of the reaction within the membrane hydrophobic phase is the same as in the aqueous cytosol, the reaction is accelerated overall because of the increased reactant concentrations within the membrane.To test this possibility, we used an electrochemical method to measure the rate of disappearance of NO in an aerobic buffered solution upon addition of various hydrophobic phases. METHODSHepatocyte Isolation and Cell Membrane Preparation. Rat hepatocytes were isolated as described (9). For membrane isolation, cells were suspended in 50 mM potassium phosphate (pH 7.4) containing 0.5 mM EDTA and sonicated (two 10-s bursts) while cooled on ice. The sonicated preparations were centrifuged at 5,000 ϫ g for 5 min at 4°C. The supernatant was subjected ...

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