Erythrocytes possess an intrinsic barrier to nitric oxide consumption

It has been reported that free hemoglobin (Hb) reacts with NO at an extremely high rate (K(Hb) approximately 10(7) M(-1) s(-1)) and that the red blood cell (RBC) membrane is highly permeable to NO. RBCs, however, react with NO 500-1000 times slower. This reduction of NO reaction rate by RBCs has been attributed to the extracellular diffusion limitation. To test whether additional limitations are also important, we designed a competition test, which allows the extracellular diffusion limitation to be distinguished from transmembrane or intracellular resistance. This test exploited the competition between free Hb and RBCs for NO generated in a homogenous phase by an NO donor. If the extracellular diffusion resistance is negligible, then the results would follow a kinetic model that assumes homogenous reaction without extracellular diffusion limitation. In this case, the measured effective reaction rate constant, K(RBC), would remain invariant of the hematocrit, extracellular-free Hb concentration, and NO donor concentration. Results show that the K(RBC) approaches a constant only when the hematocrit is greater than 10%, suggesting that at higher hematocrit, the extracellular diffusion resistance is negligible. Under such a condition, the NO consumption by RBCs is still 500-1000 times slower than that by free Hb. This result suggests that intrinsic RBC factors, such as transmembrane diffusion limitation or intracellular mechanisms, exist to reduce the NO consumption by RBCs.     

Erythrocyte consumption of nitric oxide: competition experiment and model analysis.

It is generally believed that the erythrocyte membrane is highly permeable to nitric oxide (NO). To prevent NO from freely entering and being scavenged by the red blood cell (RBC), it has been suggested that NO consumption is limited by the mass transfer resistance of the diffusion layer adjacent to the erythrocyte membrane. Recently, we (Vaughn et al. (2000). J. Biol. Chem. 275, 2342) presented an experimental technique that overcomes experimental diffusional limitations and showed that RBCs also possess a mechanism to slow nitric oxide uptake. Here, we present a mathematical analysis of this technique by modeling the NO uptake of a single cell. We obtain additional data (n = 33, total) by use of the competition experiment and, through application of the model, show that either the RBC membrane permeability to NO or the intracellular reaction rate between NO and hemoglobin (Hb) is at least 2000-fold lower than previously thought. As a result, RBCs react with NO at a rate three orders of magnitude slower than free oxyHb. This phenomena may play an important role in NO bioavailability.     

Mechanisms of slower nitric oxide uptake by red blood cells and other hemoglobin-containing vesicles

Nitric oxide (NO) acts as a smooth muscle relaxation factor and plays a crucial role in maintaining vascular homeostasis. NO is scavenged rapidly by hemoglobin (Hb). However, under normal physiological conditions, the encapsulation of Hb inside red blood cells (RBCs) significantly retards NO scavenging, permitting NO to reach the smooth muscle. The rate-limiting factors (diffusion of NO to the RBC surface, through the RBC membrane or inside of the RBC) responsible for this retardation have been the subject of much debate. Knowing the relative contribution of each of these factors is important for several reasons including optimization of the development of blood substitutes where Hb is contained within phospholipid vesicles. We have thus performed experiments of NO uptake by erythrocytes and microparticles derived from erythrocytes and conducted simulations of these data as well as that of others. We have included extracellular diffusion (that is, diffusion of the NO to the membrane) and membrane permeability, in addition to intracellular diffusion of NO, in our computational models. We find that all these mechanisms may modulate NO uptake by membrane-encapsulated Hb and that extracellular diffusion is the main rate-limiting factor for phospholipid vesicles and erythrocytes. In the case of red cell microparticles, we find a major role for membrane permeability. These results are consistent with prior studies indicating that extracellular diffusion of several gas ligands is also rate-limiting for erythrocytes, with some contribution of a low membrane permeability.     

Interactions of nitrosylhemoglobin and carboxyhemoglobin with erythrocyte.

Nitrosylhemoglobin (HbFe(II)NO) has been detected in vivo, and its role in NO transport and preservation has been discussed. To gain insight into the potential role of HbFe(II)NO, we performed in vitro experiments to determine the effect of oxygenated red blood cells (RBCs) on the dissociation of cell-free HbFe(II)NO, using carboxyhemoglobin (HbFe(II)CO) as a comparison. Results show that the apparent half-life of the cell-free HbFe(II)CO was reduced significantly in the presence of RBCs at 1% hematocrit. In contrast, RBC did not change the apparent half-life of extracellular HbFe(II)NO, but caused a shift in the HbFe(II)NO dissociation product from methemoglobin (metHbFe(III)) to oxyhemoglobin (HbFe(II)O(2)). Extracellular hemoglobin was able to extract CO from HbFe(II)CO-containing RBC, but not NO from HbFe(II)NO-containing RBC. Although these results appear to suggest some unusual interactions between HbFe(II)NO and RBC, the data are explainable by simple HbFe(II)NO dissociation and hemoglobin oxidation with known rate constants. A kinetic model consisting of these reactions shows that (i) deoxyhemoglobin is an intermediate in the reaction of HbFe(II)NO oxidation to metHbFe(III), (ii) the rate-limiting step of HbFe(II)NO decay is the dissociation of NO from HbFe(II)NO, (iii) the magnitude of NO diffusion rate constant into RBC is estimated to be approximately 10(4)M(-1)s(-1), consistent with previous results determined from a competition assay, and (iv) no additional chemical reactions are required to explain these data.     

Encapsulation of concentrated hemoglobin solution in phospholipid vesicles retards the reaction with NO, but not CO, by intracellular diffusion barrier

One physiological significance of the red blood cell (RBC) structure is that NO binding of Hb is retarded by encapsulation with the cell membrane. To clarify the mechanism, we analyzed Hb-vesicles (HbVs) with different intracellular Hb concentrations, [Hb](in), and different particle sizes using stopped-flow spectrophotometry. The apparent NO binding rate constant, k(on)('(NO)), of HbV at [Hb](in) = 1 g/dl was 2.6 x 10(7) m(-1) s(-1), which was almost equal to k(on)((NO)) of molecular Hb, indicating that the lipid membrane presents no obstacle for NO binding. With increasing [Hb](in) to 35 g/dl, k(on)('(NO)) decreased to 0.9 x 10(7) m(-1) s(-1), which was further decreased to 0.5 x 10(7) m(-1) s(-1) with enlarging particle diameter from 265 to 452 nm. For CO binding, which is intrinsically much slower than NO binding, k(on)('(CO)) did not change greatly with [Hb](in) and the particle diameter. Results obtained using diffusion simulations coupled with elementary binding reactions concur with these tendencies and clarify that NO is trapped rapidly by Hb from the interior surface region to the core of HbV at a high [Hb](in), retarding NO diffusion toward the core of HbV. In contrast, slow CO binding allows time for further CO-diffusion to the core. Simulations extrapolated to larger particles (8 mum) showing retardation even for CO binding. The obtained k(on)('(NO)) and k(on)('(NO)) yield values similar to those reported for RBCs. In summary, the intracellular, not extracellular, diffusion barrier is predominant due to the rapid NO binding that induces a rapid sink of NO from the interior surface to the core, retarding further NO diffusion and binding.     

Estimation of nitric oxide concentration in blood for different rates of generation. Evidence that intravascular nitric oxide levels are too low to exert physiological effects

Endothelium-derived nitric oxide (NO) is a potent vasodilator in the cardiovascular system. Several lines of experimental evidence suggest that NO or NO equivalents may also be generated in the blood. However, blood contains a large amount of hemoglobin (Hb) in red blood cells (RBCs). The RBC-encapsulated Hb can react very quickly with NO, which is only limited by the rate of NO diffusion into the RBCs. It is unclear what the possible NO concentration levels in blood are and how the NO diffusion coefficient (D) and the permeability (Pm) of RBC membrane to NO affect the level of NO concentration. In this study, a steady-state concentration experimental method combined with a spherical diffusion model are presented for determining D and Pm and examining the effect of NO generation rate (V0) and hematocrit (Hct) on NO concentration. It was determined that Pm is 4.5 +/- 1.5 cm/s and D is 3410 +/- 50 microm2/s at 37 degrees C. Simulations based on experimental parameters show that, when the rate of NO formation is as high as 100 nm/s, the maximal NO concentration in blood is below 0.012 nM at Pm = 4.5 cm/s and Hct = 45%. Thus, it is unlikely that NO is directly exported or generated from the RBC as an intravascular signaling molecule, because its concentration would be too low to exert a physiological role. Furthermore, our results suggest that, if RBCs export NO bioactivity, this would be through NO-derived species that can release or form NO rather than NO itself.     

Effects of the RBC membrane and increased perfusate viscosity on hypoxic pulmonary vasoconstriction.

Red blood cells (RBCs) augment hypoxic pulmonary vasoconstriction (HPV) in part by scavenging of nitric oxide (NO) by Hb (Deem S, Swenson ER, Alberts MK, Hedges RG, and Bishop MJ, Am J Respir Crit Care Med 157: 1181-1186, 1998). We studied the contribution of the RBC compartmentalization of Hb to augmentation of HPV and scavenging of NO in isolated perfused rabbit lungs. Lungs were initially perfused with buffer; HPV was provoked by a 5-min challenge with hypoxic gas (inspired O(2) fraction 0.05). Expired NO was measured continuously. Addition of free Hb to the perfusate (0.25 mg/ml) resulted in augmentation of HPV and a fall in expired NO that were similar in magnitude to those associated with a hematocrit of 30% (intracellular Hb of 100 mg/ml). Addition of dextran resulted in a blunting of HPV after free Hb but no change in expired NO. Blunting of HPV by dextran was not prevented by NO synthase inhibition with N(omega)-nitro-L-arginine and/or cyclooxygenase inhibition. RBC ghosts had a mild inhibitory effect on HPV but caused a small reduction in expired NO. In conclusion, the RBC membrane provides a barrier to NO scavenging and augmentation of HPV by Hb. Increased perfusate viscosity inhibits HPV by an undetermined mechanism.     

Thermal stability of hemoglobin crosslinked in the T-state by bis(3,5-dibromosalicyl) fumarate

Bis(3,5-dibromosalicyl) fumarate was used to crosslink oxyhemoglobin between Lys 82 beta 1 and Lys 82 beta 2 (Walder, J. A., et al. (1979) Biochemistry 18, 4265) and deoxyhemoglobin between Lys 99 alpha 1 and Lys 99 alpha 2 (Chatterjee R.Y., et al. (1986) J. Biol. Chem. 261, 9929). Thermal denaturations demonstrated that alpha crosslinked hemoglobin (alpha 99XLHb A) has the same stability as the beta crosslinked one (beta 82XLHb A). Both alpha and beta crosslinked methemoglobins have a denaturation temperature in 0.9 M guanidine of 57 degrees C compared to 41 degrees C of Hb A. The second product from the T-state crosslinking reaction was found to be crosslinked between the beta chains by chain separation and amino acid analysis. The possible positions for this crosslink are limited to the bisphosphoglycerate binding site in the three-dimensional structure. Its stability is comparable to that of the alpha 99XLHb A or beta 82XLHb A. These modified hemoglobins are potential blood substitutes.     

Erythrocyte nitric oxide transport reduced by a submembrane cytoskeletal barrier

Encapsulation of hemoglobin (Hb) within red blood cells (RBCs) preserves nitric oxide (NO) bioactivity. With encapsulation, millimolar concentrations of Hb quench only a fraction of NO bioactivity, whereas mere micromolar concentrations of cell-free Hb completely quench NO bioactivity. A submembrane cytoskeletal barrier has been hypothesized to account for the lowered quenching of NO bioactivity. In order to substantiate this hypothesis, here, the underlying submembrane cytoskeletal barrier was physically reduced and the rate of NO entry into the modified RBC measured. The submembrane cytoskeletal barrier of normal and depleted RBCs was characterized using atomic force microscopy and the lipid to protein ratio measured. The reduction in the submembrane cytoskeletal barrier resulted in an increase in the rate of NO entry. We suggest that the underlying submembrane cytoskeleton may be a key component of RBC mediated regulation of NO bioavailability.     

Nitric oxide red blood cell membrane permeability at high and low oxygen tension.

Red blood cell (RBC) encapsulated hemoglobin in the blood scavenges nitric oxide (NO) much more slowly than cell-free hemoglobin would. Part of this reduced NO scavenging has been attributed to an intrinsic membrane barrier to diffusion of NO through the RBC membrane. Published values for the permeability of RBCs to NO vary over several orders of magnitude. Recently, the rate that RBCs scavenge NO has been shown to depend on the hematocrit (percentage volume of RBCs) and oxygen tension. The difference in rate constants was hypothesized to be due to oxygen modulation of the RBC membrane permeability, but also could have been due to the difference in bimolecular rate constants for the reaction of NO and oxygenated vs deoxygenated hemoglobin. Here, we model NO scavenging by RBCs under previously published experimental conditions. A finite-element based computer program model is constrained by published values for the reaction rates of NO with oxygenated and deoxygenated hemoglobin as well as RBC NO scavenging rates. We find that the permeability of RBCs to NO under oxygenated conditions is between 4400 and 5100 microm s(-1) while the permeability under deoxygenated conditions is greater than 64,000 microm s(-1). The permeability changes by a factor of 10 or more upon oxygenation of anoxic RBCs. These results may have important implications with respect to NO import or export in physiology.     

Simultaneous reconstitution of Escherichia coli membrane vesicles with D-lactate and D-amino acid dehydrogenases

Purified preparations of D-amino acid dehydrogenase [Olsiewski, P.J., Kaczorowski, G. J., & Walsh, C. T. (1980) J. Biol. Chem. 225, 4487] and D-lactate dehydrogenase [Kohn, L.D., & Kaback, H.R. (1973) J. Biol. Chem. 248, 7012] bind independently to right-side-out and inverted Escherichia coli vesicles and to phosphatidylcholine liposomes without detectable competition. The reconstituted vesicles catalyze D-lactate- and D-alanine-dependent respiration (O2 uptake), proton translocation, and proton/lactose symport. The enzymes do not share common sites of association on either face of the E. coli membrane, and binding of both enzymes to the bilayer appears to be due to nonspecific affinity for the surface rather than specific binding to proteinaceous receptors. Each enzyme, however, appears to reduce a common proton translocating step in the membrane-bound respiratory chain, and substrate-derived electrons are transferred through a common rate-determining redox component that precedes the site of proton translocation. The results suggest that although binding is nonspecific, there is a common site for proton translocation in the membrane between the flavin-linked dehydrogenases and the cytochromes and that this site is accessible by distinct routes of electron transfer from primary dehydrogenases on either surface of the membrane.     

Mechanism of oxidative damage to fish red blood cells by ozone

The present study was conducted to elucidate the adverse effects of ozone exposure on rainbow trout (Oncorhynchus mykiss) red blood cells (RBCs). We evaluated whether hemoglobin (Hb) or Hb-derived free iron could participate in the RBC damage using an in vitro ozone exposure system. Ozone exposure induced hemolysis, formation of methemoglobin, and RBC membrane lipid peroxidation. This RBC damage was not suppressed by the addition of a specific iron chelator (deferoxamine mesilate) to the medium but was suppressed by carbon monoxide (CO) treatment before ozone exposure. Generation of hydrogen peroxide (H2O2) in RBC was observed upon ozone exposure but was significantly suppressed by CO treatment before ozone exposure. Thus the Hb status (i.e., Hb redox condition) and H2O2 generation in RBC should play important roles in mediating RBC damage by ozone exposure. In other words, neither ozone nor its derivative directly attacked from the outside of the cell, but ozone that penetrated through the membrane derived the reactive oxygen species from Hb inside of the cell.     

Generating S-Nitrosothiols from Hemoglobin: MECHANISMS,CONFORMATIONAL DEPENDENCE,AND PHYSIOLOGICAL RELEVANCE*

In vitro, ferrous deoxy-hemes in hemoglobin (Hb) react with nitrite to generate nitric oxide (NO) through a nitrite reductase reaction. In vivo studies indicate Hb with nitrite can be a source of NO bioactivity. The nitrite reductase reaction does not appear to account fully for this activity because free NO is short lived especially within the red blood cell. Thus, the exporting of NO bioactivity both out of the RBC and over a large distance requires an additional mechanism. A nitrite anhydrase (NA) reaction in which N₂O₃, a potent S-nitrosating agent, is produced through the reaction of NO with ferric heme-bound nitrite has been proposed (Basu, S., Grubina, R., Huang, J., Conradie, J., Huang, Z., Jeffers, A., Jiang, A., He, X., Azarov, I., Seibert, R., Mehta, A., Patel, R., King, S. B., Hogg, N., Ghosh, A., Gladwin, M. T., and Kim-Shapiro, D. B. (2007) Nat. Chem. Biol. 3, 785–794) as a possible mechanism. Legitimate concerns, including physiological relevance and the nature of the mechanism, have been raised concerning the NA reaction. This study addresses these concerns demonstrating NO and nitrite with ferric hemes under near physiological conditions yield an intermediate having the properties of the purported NA heme-bound N₂O₃ intermediate. The results indicate that ferric heme sites, traditionally viewed as a source of potential toxicity, can be functionally significant, especially for partially oxygenated/partially met-R state Hb that arises from the NO dioxygenation reaction. In the presence of low levels of nitrite and either NO or a suitable reductant such as l-cysteine, these ferric heme sites can function as a generator for the formation of S-nitrosothiols such as S-nitrosoglutathione and, as such, should be considered as a source of RBC-derived and exportable bioactive NO.     

Copper-dependent autocleavage of glypican-1 heparan sulfate by nitric oxide derived from intrinsic nitrosothiols

Cell surface heparan sulfate proteoglycans facilitate uptake of growth-promoting polyamines (Belting, M., Borsig, L., Fuster, M. M., Brown, J. R., Persson, L., Fransson, L.-A., and Esko, J. D. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 371-376). Increased polyamine uptake correlates with an increased number of positively charged N-unsubstituted glucosamine units in the otherwise polyanionic heparan sulfate chains of glypican-1. During intracellular recycling of glypican-1, there is an NO-dependent deaminative cleavage of heparan sulfate at these glucosamine units, which would eliminate the positive charges (Ding, K., Sandgren, S., Mani, K., Belting, M., and Fransson, L.-A. (2001) J. Biol. Chem. 276, 46779-46791). Here, using both biochemical and microscopic techniques, we have identified and isolated S-nitrosylated forms of glypican-1 as well as slightly charged glypican-1 glycoforms containing heparan sulfate chains rich in N-unsubstituted glucosamines. These glycoforms were converted to highly charged species upon treatment of cells with 1 mm l-ascorbate, which releases NO from nitrosothiols, resulting in deaminative cleavage of heparan sulfate at the N-unsubstituted glucosamines. S-Nitrosylation and subsequent deaminative cleavage were abrogated by inhibition of a Cu(2+)/Cu(+) redox cycle. Under cell-free conditions, purified S-nitrosylated glypican-1 was able to autocleave its heparan sulfate chains when NO release was triggered by l-ascorbate. The heparan sulfate fragments generated in cells during this autocatalytic process contained terminal anhydromannose residues. We conclude that the core protein of glypican-1 can slowly accumulate NO as nitrosothiols, whereas Cu(2+) is reduced to Cu(+). Subsequent release of NO results in efficient deaminative cleavage of the heparan sulfate chains attached to the same core protein, whereas Cu(+) is oxidized to Cu(2+).     

Redefining the facilitated transport of mannose in human cells: absence of a glucose-insensitive, high-affinity facilitated mannose transport system

Current evidence suggests that extracellular mannose can be transported intracellularly and utilized for glycoprotein synthesis; however, the identity and the functional characteristics of the transporters of mannose are controversial. Although the glucose transporters are capable of transporting mannose, it has been postulated that the entry of mannose in mammalian cells is mediated by a transporter that is insensitive to glucose [Panneerselvam, K., and Freeze, H. (1996) J. Biol. Chem. 271, 9417-9421] or by a transporter induced by cell treatment with metformin [Shang, J., and Lehrman, M. A. (2004) J. Biol. Chem. 279, 9703-9712]. We performed a detailed analysis of the uptake of mannose in normal human erythrocytes and in leukemia cell line HL-60. Short uptake assays allowed the identification of a single functional activity involved in mannose uptake in both cell types, with a K(m) for transport of 6 mM. Transport was inhibited in a competitive manner by classical glucose transporter substrates. Similarly, the glucose transporter inhibitors cytochalasin B, genistein, and myricetin inhibited mannose transport by 100%. Using long uptake experiments, we identified a second, high-affinity component associated with the intracellular trapping of mannose in the HL-60 cells that is not directly involved in the transport of mannose via the glucose transporters. Thus, the transport of mannose via glucose transporters is a process which is kinetically and biologically separable from its intracellular trapping. A general survey of human cells revealed that mannose uptake was entirely blocked by concentrations of cytochalasin B that obliterates the activity of the glucose transporters. The transport and inhibition data demonstrate that extracellular mannose, whose physiological concentration is in the micromolar range, enters cells in the presence of physiological concentrations of glucose. Overall, our data indicate that transport through the glucose transporter is the main mechanism by which human cells acquire mannose.     

The kinetic and isotopic competence of nitric oxide as an intermediate in denitrification

Rates of NO uptake by five denitrifying bacteria were estimated by NO-electrode and gas chromatography methods under conditions of rather low cell densities and [NOaq]. The rates so measured, VmaxNO, represent lower limits for the true value of that parameter, but nevertheless exceed Vmax for nitrite uptake, VmaxNi, by a factor of two typically. Previous estimates under suboptimal conditions had placed VmaxNO at 0.3-0.5 of VmaxNi (St. John, R. T., and Hollocher, T. C. (1977) J. Biol. Chem. 252, 212-218; Garber, E. A. E., and Hollocher, T.C. (1981) J. Biol. Chem. 256, 5459-5465). The steady-state [NOaq] during denitrification of nitrite by nitrate-grown cells was less than or equal to 1 microM. The above observations, taken with a recent direct estimate for the KmNO for NO uptake of 0.4 microM (Zafiriou, O. C., Hanley, Q. S., and Snyder, G. (1989) J. Biol. Chem. 264, 5694-5699), would allow NO to be a free intermediate between nitrite and N2O with steady-state concentrations of less than or equal to 0.4 microM. As the result of special conditions during cell growth or differential inhibition by azide, it was possible to establish systems that accumulated NO during denitrification of nitrite. In all such cases, VmaxNO less than VmaxNi, and the time required to reach the maximum [NOaq] corresponded closely to the time needed to exhaust the nitrite. A semiquantitative isotope experiment with Paracoccus denitrificans demonstrated the trapping of 15NO from 15NO2- in a pool of NOaq. A quantitative isotope method using low densities of the same bacterium showed that 15N from 15NO2- and 14N from NOg combine randomly to form N2O during the simultaneous denitrification of 15NO2- and NO. The result requires that the pathways from nitrite and NO share a common mononitrogen intermediate. Results to the contrary obtained at high cell densities (first two references cited above) are now believed to have been due to technical artifacts. The present results are consistent with the view that NO is under kinetic control as a free intermediate in denitrification and serve to remove previously imagined constraints on this view.     

Lysophospholipids open the two-pore domain mechano-gated K(+) channels TREK-1 and TRAAK

The two-pore (2P) domain K(+) channels TREK-1 and TRAAK are opened by membrane stretch as well as arachidonic acid (AA) (Patel, A. J., Honoré, E., Maingret, F., Lesage, F., Fink, M., Duprat, F., and Lazdunski, M. (1998) EMBO J. 17, 4283-4290; Maingret, F., Patel, A. J., Lesage, F., Lazdunski, M., and Honoré, E. (1999) J. Biol. Chem. 274, 26691-26696; Maingret, F., Fosset, M., Lesage, F., Lazdunski, M. , and Honoré, E. (1999) J. Biol. Chem. 274, 1381-1387. We demonstrate that lysophospholipids (LPs) and platelet-activating factor also produce large specific and reversible activations of TREK-1 and TRAAK. LPs activation is a function of the size of the polar head and length of the acyl chain but is independent of the charge of the molecule. Bath application of lysophosphatidylcholine (LPC) immediately opens TREK-1 and TRAAK in the cell-attached patch configuration. In excised patches, LPC activation is lost, whereas AA still produces maximal opening. The carboxyl-terminal region of TREK-1, but not the amino terminus and the extracellular loop M1P1, is critically required for LPC activation. LPC activation is indirect and may possibly involve a cytosolic factor, whereas AA directly interacts with either the channel proteins or the bilayer and mimics stretch. Opening of TREK-1 and TRAAK by fatty acids and LPs may be an important switch in the regulation of synaptic function and may also play a protective role during ischemia and inflammation.     

Mechanism of superoxide generation by neuronal nitric-oxide synthase

Neuronal nitric-oxide synthase (NOS I) in the absence of L-arginine has previously been shown to generate superoxide (O-2) (Pou, S., Pou, W. S., Bredt, D. S., Snyder, S. H., and Rosen, G. M. (1992) J. Biol. Chem. 267, 24173-24176). In the presence of L-arginine, NOS I produces nitric oxide (NO.). Yet the competition between O2 and L-arginine for electrons, and by implication formation of O-2, has until recently remained undefined. Herein, we investigated this relationship, observing O-2 generation even at saturating levels of L-arginine. Of interest was the finding that the frequently used NOS inhibitor NG-monomethyl L-arginine enhanced O-2 production in the presence of L-arginine because this antagonist attenuated NO. formation. Whereas diphenyliodonium chloride inhibited O-2, blockers of heme such as NaCN, 1-phenylimidazole, and imidazole likewise prevented the formation of O-2 at concentrations that inhibited NO. formation from L-arginine. Taken together these data demonstrate that NOS I generates O-2 and the formation of this free radical occurs at the heme domain.     

Dissociation of dimers of human hemoglobins A and F into monomers

Dissociation of alpha beta and alpha gamma dimers of human hemoglobins (Hb) A and F into monomers was studied by alpha chain exchange (Shaeffer, J. R., McDonald, M. J., Turci, S. M., Dinda, D. M., and Bunn, H. F. (1984) J. Biol. Chem. 259, 14544-14547). Unlabeled carbonmonoxy-Hb A was incubated with trace amounts of preparatively purified, native, 3H-alpha subunits in 10 mM sodium phosphate, pH 7.0, at 25 degrees C. At appropriate times, free alpha monomers were separated from Hb A tetramers by anion exchange high performance liquid chromatography. Transfer of radioactivity from the alpha chain pool into Hb A was measured, yielding a first order dimer dissociation rate constant, k2 = (3.2 +/- 0.3) X 10(-3) h-1. The Arrhenius plot of k2 was linear between 7 and 37 degrees C, yielding an enthalpy of activation of 23 kcal/alpha beta dimer. As the chloride concentration was raised from 0 to 0.2 M, the dissociation rate increased 3-fold; with higher salt concentrations, however, the rate gradually returned to baseline. This rate was not altered by raising the pH from 6.5 to 7.2, but as pH was further raised to 8.4, kappa 2 increased about 3-fold. Hb F, which has an increased stability at alkaline pH, dissociated into alpha and gamma monomers 3 times more slowly than Hb A. Moreover, the dimer-monomer dissociation of Hb F was characterized by a significantly reduced pH dependence. These results demonstrate that both alpha beta and alpha gamma dimers of Hb A and Hb F dissociate reversibly into monomers under physiologic conditions. The differential pH dependence for dimer dissociation between Hb A and Hb F suggests that specific amino acid replacement at the alpha 1 gamma 1 interface confers increased resistance to alkaline denaturation.