Cooperative effect of inositol hexakisphosphate, bezafibrate, and clofibric acid on the spectroscopic properties of the nitric oxide derivative of ferrous human hemoglobin

The cooperative effect of inositol hexakisphosphate (IHP), bezafibrate (BZF), and clofibric acid (CFA) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous human hemoglobin (HbNO) has been investigated quantitatively. In the presence of IHP, BZF, and CFA, the X-band EPR spectra and the absorption spectra in the Soret region of HbNO display the same basic characteristics described in the presence of 2,3-diphosphoglycerate (2,3-DPG), which have been attributed to a low affinity conformation of the tetramer. Addition to HbNO of two allosteric effectors together (such as IHP and BZF, or IHP and CFA) further stabilizes the low affinity conformation of the ligated hemoprotein (i.e., HbNO). Moreover, in the presence of saturating amounts of IHP, the affinity of BZF and CFA for HbNO increases by about fifteenfold. Likewise, in the presence of both IHP and BZF, as well as in IHP and CFA, the oxygen affinity for ferrous human hemoglobin (Hb) is reduced with respect to that observed in the presence of IHP, BZF, or CFA alone, which in turn is lower than that reported in the absence of any allosteric effector. All the data were obtained at pH 7.0 (in 1.0 × 10⁻¹ M N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]/NaOH buffer system plus 1.0 × 10⁻¹ M NaCl), as well as at 100 K and/or 20°C. The results here reported represent clearcut evidence for the cooperative and specific (i.e., functionally relevant) binding of IHP, BZF, and CFA to Hb.     

Effect of bezafibrate and clofibric acid on the spectroscopic properties of the nitric oxide derivative of ferrous human hemoglobin.

The effect of bezafibrate (BZF) and clofibric acid (CFA) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous human hemoglobin (HbNO) has been investigated quantitatively. In the presence of BZF and CFA, the X-band EPR spectra and the absorption spectra in the Soret region of HbNO display the same basic characteristics described in the presence of inositol hexakisphosphate (IHP) and 2, 3-diphosphoglycerate (2,3-DPG). Next, in the presence of these allosteric effectors, the oxygen affinity for ferrous human hemoglobin (Hb) is reduced. These findings indicate that BZF and CFA, as already reported for IHP and 2, 3-DPG, induce the stabilization of a low affinity conformation of the ligated hemoprotein (i.e., HbNO). Values of the apparent equilibrium constant for BZF and CFA binding to HbNO (K) are 1.5(+/- 0.2) x 10(-2) M and 2.8(+/- 0.3) x 10(-2) M, respectively, at pH 7.0 (in 0.1 M N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]/NaOH buffer system plus 0.1 M NaCl) and 20 degrees C. The results reported here represent clearcut evidence for BZF and CFA specific (i.e., functionally relevant) binding to a ligated derivative of Hb (i.e., HbNO).     

Different structural effects of allosteric modulators on subunits of tetrameric ferrous nitrosylated human hemoglobin: an EPR spectroscopic study

 The X-band EPR spectroscopic features of the ferrous nitrosylated derivative of α(Fe)₂β(Co)₂ and of α(Co)₂β(Fe)₂ metal hybrids of human hemoglobin (Hb) have been investigated at pH 7.0 and analyzed in parallel with those of the native nitrosylated tetramer (HbNO). The effect of 2,3-biphosphoglycerate (BPG), inositol hexakisphosphate (IHP) and bezafibrate (BZF) has been investigated in order to understand the perturbations induced on α and β subunits in the tetramer by the binding of allosteric effectors. A large perturbation is observed in both subunits upon BZF binding, while in the case of IHP only α-chains are affected; on the other hand, BPG leaves both chains essentially unperturbed. Thus, different binding modes of allosteric effectors to HbNO may occur, and the simultaneous addition of two effector molecules, namely BPG and BZF or IHP and BZF to HbNO, brings about different alterations of the X-band EPR spectroscopic properties. This behavior indicates that the intramolecular communication pathway(s) between the heme and the binding pockets of the heterotropic ligands (i.e., IHP and BZF, or BPG and BZF) are different, leading to distinct structural perturbations. Received: 19 September 1997 / Accepted: 16 December 1997     

Decay of individual Escherichia coli trp messenger RNA molecules is sequentially ordered.

Human fluoromethaemoglobin with inositol hexaphosphate (IHP) in 0.05 m-phosphate buffer was crystallized by addition of polyethylene glycol (PEG). The crystals are isomorphous with those of deoxyhaemoglobin A without IHP grown in solutions containing PEG by Ward et al. (1975). The structure was investigated by means of a difference Fourier synthesis against deoxyhaemoglobin A based on X-ray data collected within a limiting sphere of 3.5 Å^?1. The four subunits are arranged in the quaternary T structure and IHP is bound at the same site between the β chains as in deoxyhaemoglobin. In both the α and β haem regions the distance between the haem plane and the F helix is reduced in fluoromethaemoglobin relative to deoxyhaemoglobin and the iron atom is moved from the proximal towards the distal side of the plane, but the change, if any, in the distance between the iron and the N_ε of the proximal histidine cannot be clearly established. The α Fe in fluoromethaemoglobin is either in the haem plane or up to 0.8 Å on the distal side, suggesting the possibility of rupture of the bond to the histidines N_ε; it was not possible to estimate the position of the β iron. The main spectral changes associated with the reaction of fluoromethaemoglobin with IHP take place in less than 3 ms at room temperature.     

Temperature dependence of Q-band electron paramagnetic resonance spectra of nitrosyl heme proteins

The Q-band (35 GHz) electron paramagnetic resonance (EPR) spectra of nitrosyl hemoglobin (HbNO) and nitrosyl myoglobin (MbNO) were studied as a function of temperature between 19 K and 200 K. The spectra of both heme proteins show two classes of variations as a function of temperature. The first one has previously been associated with the existence of two paramagnetic species, one with rhombic and the other with axial symmetry. The second one manifests itself in changes in the g-factors and linewidths of each species. These changes are correlated with the conformational substates model and associate the variations of g-values with changes in the angle of the N(his)-Fe-N(NO) bond in the rhombic species and with changes in the distance between Fe and N of the proximal (F8) histidine in the axial species.     

Influence of globin structures on the state of the heme. Ferrous low spin derivatives.

Studies of high spin ferrous and ferric derivatives led us to conclude that in the quaternary R structure the state of the hemes is similar to that in the free alpha and beta subunits, but in the T structure a tension acts on the hemes which tries to pull the iron and the proximal histidine further from the plane of the porphyrin. We have now studied the effect of inositol hexaphosphate (IHP) on the three low spin ferrous compounds of hemoglobin with O2, CO, and NO. IHP failed to switch the quaternary structure of carbonmonoxy- and oxyhemoglobin A to the T state, but merely caused a transition to an as yet undefined modification of the R structure. IHP is known to cause a switch to the T structure in hemoglobin Kansas. We have found that this switch induces red shifts of the visible alpha and beta absorption bands and the appearance of a shoulder on the red side of the alpha band; these changes are very weak in carbonmonoxy- and slightly stronger in oxyhemoglobin Kansas. As already noted by previous authors, addition of IHP to nitrosylhemoglobin A induces all the changes in uv absorption and CD spectra, sulfhydryl reactivities, and exchangeable proton resonances normally associated with the R leads to T transition, and is accompanied by large changes in the Soret and visible absorption bands. Experiments with nitrosyl hybrids show that these changes in absorption are caused predominantly by the hemes in the alpha subunits. In the accompanying paper Maxwell and Caughey (J. C. Maxwell and W. S. Caughey (1976), Biochemistry, following paper in this issue) report that the NO in nitrosylhemoglobin without IHP gives a single ir stretching frequency characteristic for six-coordinated nitrosyl hemes; addition of IHP causes the appearance of a second ir band, of intensity equal to that of the first, which is characteristic for five-coordinated nitrosyl hemes. Taken together, these results show that the R leads to T transition causes either a rupture or at least a very dramatic stretching of the bond from the iron to the heme-linked histidine, such that an equilibrium is set up between five- and six-coordinated hemes, biased toward five-coordinated hemes in the alpha and six-coordinated ones in the beta subunits. The reason why IHP can switch nitrosyl-, but not carbonmonoxy- or oxyhemoglobin A, from the R to the T structure is to be found in the weakening of the iron-histidine bond by the unpaired NO electron and by the very short Fe-NO bond length.     

Spectroscopic studies of protein-heme interactions accompanying the allosteric transition in methemoglobins

Resonance Raman, optical absorption, and circular dichroism spectroscopic techniques have been used to examine the effect of the addition of inositol hexaphosphate (IHP) to a series of carp and human methemoglobin derivatives. Markers of spin equilibrium in the high-frequency region (1450-1650 cm-1) of the resonance Raman spectrum yield high/low-spin ratios consistent with direct magnetic susceptibility measurements. Changes in the low-frequency region (100-600 cm-1) of the resonance Raman spectrum appear to correlate with the quaternary structure transition. Changes in the ultraviolet absorption spectra and the circular dichroism spectra also appear to be related to the quaternary structure change. By using the resonance Raman spin markers, we find that those derivatives of carp methemoglobin which are in spin equilibrium have a larger ratio of high-spin to low-spin populations than the corresponding derivatives of human methemoglobin. Upon the addition of IHP to the methemoglobins the spin equilibrium is shifted toward a larger high-spin population. This change in equilibrium is larger for the carp protein than for the human protein. We obtain an IHP-induced change in the free energy difference between the high-spin and low-spin states of 300 cal/mol for those human methemoglobins in which a quaternary structure change occurs and 600 cal/mol for carp methemoglobins. Our data are consistent with a quaternary structure change induced by IHP in all the carp methemoglobins studied (F-, H2O, SCN-, NO2-, N3-, and CN-) and in the F-, H2O, and SCN- derivatives of the human protein but not in the NO2-, N3-, and CN- derivatives. The Fe-CN stretching mode has been identified by isotopic substitution and found to be unchanged in frequency in carp CN- metHb when the quaternary structure is changed. On the basis of our results we conclude that the protein forces at the heme due to the addition of IHP do not significantly affect the position of the iron atom with respect to the heme plane. Rather, the changes in spin equilibrium may be caused by protein-induced changes in the orientation of the proximal histidine or tertiary structure changes in the heme pocket which affect the porphyrin macrocycle. Either of these changes, or a combination thereof, leads to changes in the iron d orbital energies and concomitant changes in the spin equilibrium.     

Enthalpy changes for inositol hexaphosphate binding to hemoglobins A and M Iwate.

Enthalpies of inositol hexaphosphate (IHP) binding to deoxy and carbonmonoxy (CO) HbA and HbM Iwate have been determined calorimetrically and compared as functions of pH. Values for deoxy HbA and for deoxy HbM Iwate are similar with CO HbM Iwate yielding slightly less heat of reaction. The results support the existence of both deoxy and CO HbM Iwate in T-like structures with only minor modifications occurring upon CO binding. For HbA observed heats of IHP binding have been corrected for heats of extraction of reacting protons from buffer. The resulting intrinsic IHP binding enthalpies show consistent values of ?7 to ?11 kcal/mol proton absorbed in binding. We suggest that a major driving force for organic phosphate binding is the exothermic protonation of histidine and/or a α-amino nitrogens induced by proximity of phosphate negative charges.     

Detection of nitric oxide production in lipopolysaccharide-treated rats by ESR using carbon monoxide hemoglobin.

Release of nitric oxide (NO), from macrophages activated with E. coli lipopolysaccharide (LPS) and endothelial cells, has been proposed using chemiluminescence and spectrophotometry. However these methods can not distinguish NO from NO2-. The present study was aimed to prove in vivo production of NO, by ESR using CO-hemoglobin (HbCO) as a trapping agent of NO in the peritoneal cavity of rats treated with LPS. We detected a broad signal in the recovered HbCO solution. Inositol hexaphosphate induced a three-line hyperfine structure, characteristic of NO-hemoglobin (HbNO). In the arterial blood, ESR signal of HbNO with faint hyperfine structure was detected. NG-Monomethyl-L-arginine inhibited the formation of HbNO. HbNO was not detected in the peritoneal cavity of the LPS-untreated rat given i.p. both NO2- and HbCO. HbNO was, therefore, derived from NO, not from NO2-. These results show that free NO is produced in vivo by the stimulation of LPS.     

Functional consequences of the creation of an Asp-His-Fe triad in a 3/3 globin

The proximal side of dehaloperoxidase-hemoglobin A (DHP A) from Amphitrite ornata has been modified via site-directed mutagenesis of methionine 86 into aspartate (M86D) to introduce an Asp-His-Fe triad charge relay. X-ray crystallographic structure determination of the metcyano forms of M86D [Protein Data Bank (PDB) entry 3MYN ] and M86E (PDB entry 3MYM ) mutants reveal the structural origins of a stable catalytic triad in DHP A. A decrease in the rate of H(2)O(2) activation as well as a lowered reduction potential versus that of the wild-type enzyme was observed in M86D. One possible explanation for the significantly lower activity is an increased affinity for the distal histidine in binding to the heme Fe to form a bis-histidine adduct. Resonance Raman spectroscopy demonstrates a pH-dependent ligation by the distal histidine in M86D, which is indicative of an increased trans effect. At pH 5.0, the heme Fe is five-coordinate, and this structure resembles the wild-type DHP A resting state. However, at pH 7.0, the distal histidine appears to form a six-coordinate ferric bis-histidine (hemichrome) adduct. These observations can be explained by the effect of the increased positive charge on the heme Fe on the formation of a six-coordinate low-spin adduct, which inhibits the ligation and activation of H(2)O(2) as required for peroxidase activity. The results suggest that the proximal charge relay in peroxidases regulate the redox potential of the heme Fe but that the trans effect is a carefully balanced property that can both activate H(2)O(2) and attract ligation by the distal histidine. To understand the balance of forces that modulate peroxidase reactivity, we studied three M86 mutants, M86A, M86D, and M86E, by spectroelectrochemistry and nuclear magnetic resonance spectroscopy of (13)C- and (15)N-labeled cyanide adducts as probes of the redox potential and of the trans effect in the heme Fe, both of which can be correlated with the proximity of negative charge to the N(δ) hydrogen of the proximal histidine, consistent with an Asp-His-Fe charge relay observed in heme peroxidases.     

Oxygen and CO binding to triply NO and asymmetric NO/CO hemoglobin hybrids.

The bimolecular and geminate CO recombination kinetics have been measured for hemoglobin (Hb) with over 90% of the ligand binding sites occupied by NO. Since Hb(NO)4 with inositol hexaphosphate (IHP) at pH below 7 is thought to take on the low affinity (deoxy) conformation, the goal of the experiments was to determine whether the species IHPHb-(NO)3(CO) also exists in this quaternary structure, which would allow ligand binding studies to tetramers in the deoxy conformation. For samples at pH 6.6 in the presence of IHP, the bimolecular kinetics show only a slow phase with rate 7 x 10(4) M-1 s-1, characteristic of CO binding to deoxy Hb, indicating that the triply NO tetramers are in the deoxy conformation. Unlike Hb(CO)4, the fraction recombination occurring during the geminate phase is low (< 1%) in aqueous solutions, suggesting that the IHPHb(NO)3(CO) hybrid is also essentially in the deoxy conformation. By mixing stock solutions of HbCO and HbNO, the initial exchange of dimers produces asymmetric (alpha NO beta NO/alpha CO beta CO) hybrids. At low pH in the presence of IHP, this hybrid also displays a high bimolecular quantum yield and a large fraction of slow (deoxy-like) CO recombination; the slow bimolecular kinetics show components of equal amplitude with rates 7 and 20 x 10(4) M-1 s-1, probably reflecting the differences in the alpha and beta chains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton-linked subunit heterogeneity in ferrous nitrosylated human adult hemoglobin: an EPR study

The effect of pH on the X-band electron paramagnetic resonance (EPR) spectrum of ferrous nitrosylated human adult tetrameric hemoglobin (HbNO) as well as of ferrous nitrosylated monomeric alpha- and beta-chains has been investigated, at -163 degrees C. At pH 7.3, the X-band EPR spectrum of tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains displays a rhombic shape. Lowering the pH from 7.3 to 3.0, tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains undergo a transition towards a species characterized by a X-band EPR spectrum with a three-line splitting centered at 334mT. These pH-dependent spectroscopic changes may be taken as indicative of the cleavage, or the severe weakening, of the proximal HisF8-Fe bond. In tetrameric HbNO, the pH-dependent spectroscopic changes depend on the acid-base equilibrium of two apparent ionizing groups with pK(a) values of 5.8 and 3.8. By contrast, the pH-dependent spectroscopic changes occurring in ferrous nitrosylated monomeric alpha- and beta-chains depend on the acid-base equilibrium of one apparent ionizing group with pK(a) values of 4.8 and 4.7, respectively. The different pK(a) values for the proton-linked spectroscopic transition(s) of tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains suggest that the quaternary assembly drastically affects the strength of the proximal HisF8-Fe bond in both subunits. This probably reflects a 'quaternary effect', i.e., structural changes in both subunits upon tetrameric assembly, which is associated to a relevant variation of functional properties (i.e., proton affinity).     

Effect of removal of a salt-bridge on the oxygen binding properties and the electronic structure of heme in cobalt-iron hybrid hemoglobin.

In order to clarify the role of salt-bridges in hemoglobin, the oxygen equilibrium curves and electron paramagnetic resonance (EPR) spectra of cobalt-iron hybrid hemoglobins were determined. The EPR spectra of deoxy alpha(Co)2 beta(Fe)2 could be interpreted as a mixture of two distinct paramagnetic species: one showed a maximum of the first derivative spectrum at g = 2.39 and the other at g = 2.33. The oxygen equilibrium curves of the hybrid indicated that the former is assignable to the T structure and the latter to the R structure. The cooperativity of oxygen binding of alpha(Co)2 beta(Fe)2 exhibited a maximum at g = 2.33, which is characteristic of the R structure, regardless of the pH. Addition of inositol hexaphosphate (IHP) to des-Arg alpha(Co)2 beta(Fe)2 restored the cooperativity of oxygen binding, which implies that the deoxygenated form of des-Arg alpha(Co)2 beta(Fe)2 is converted to the T structure upon addition of IHP. However, the EPR signal at g = 2.39 was not restored upon conversion to the T structure by addition of IHP. It is therefore concluded that the EPR spectrum of the deoxy alpha(Co) subunit depends both on the quaternary structure and on the localized strain at the heme.     

Resonance Raman studies indicate a unique heme active site in prostaglandin H synthase

Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and -2) catalyze the first two steps in the biosynthesis of prostaglandins. Resonance Raman spectroscopy was used to characterize the PGHS heme active site and its immediate environment. Ferric PGHS-1 has a predominant six-coordinate high-spin heme at room temperature, with water as the sixth ligand. The proximal histidine ligand (or the distal water ligand) of this hexacoordinate high-spin heme species was reversibly photolabile, leading to a pentacoordinate high-spin ferric heme iron. Ferrous PGHS-1 has a single species of five-coordinate high-spin heme, as evident from nu(2) at 1558 cm(-1) and nu(3) at 1471 cm(-1). nu(4) at 1359 cm(-1) indicates that histidine is the proximal ligand. A weak band at 226-228 cm(-1) was tentatively assigned as the Fe-His stretching vibration. Cyanoferric PGHS-1 exhibited a nu(Fe)(-)(CN) line at 446 cm(-1) and delta(Fe)(-)(C)(-)(N) at 410 cm(-1), indicating a "linear" Fe-C-N binding conformation with the proximal histidine. This linkage agrees well with the open distal heme pocket in PGHS-1. The ferrous PGHS-1 CO complex exhibited three important marker lines: nu(Fe)(-)(CO) (531 cm(-1)), delta(Fe)(-)(C)(-)(O) (567 cm(-1)), and nu(C)(-)(O) (1954 cm(-1)). No hydrogen bonding was detected for the heme-bound CO in PGHS-1. These frequencies markedly deviated from the nu(Fe)(-)(CO)/nu(C)(-)(O) correlation curve for heme proteins and porphyrins with a proximal histidine or imidazolate, suggesting an extremely weak bond between the heme iron and the proximal histidine in PGHS-1. At alkaline pH, PGHS-1 is converted to a second CO binding conformation (nu(Fe)(-)(CO): 496 cm(-1)) where disruption of the hydrogen bonding interactions to the proximal histidine may occur.     

Electrochemical assay for determining nitrosyl derivatives of human hemoglobin: nitrosylhemoglobin and S-nitrosylhemoglobin

Nitric oxide (NO) is an important biological regulator. It can bind to heme iron and form NO+, involved in the synthesis of S-nitrosothiols (-SNOs). NO reacts with human hemoglobin (Hb) to produce the derivatives: S-nitrosylhemoglobin (-SNOHb) and nitrosylhemoglobin (HbNO). At neutral pH values, free NO does not react directly with the -SH groups of Hb. The reductive nitrosylation of Fe(III) heme upon reaction with NO has long been studied, but it is not yet completely known. To quantify the reaction of NO with Hb, we developed a new, sensitive (nanomolar concentration range) electrochemical assay to selectively measure HbNO and -SNOHb. The assay also allows the monitoring of free NO during the reaction with human Fe(III)Hb and Fe(II)HbO(2).     

Differences in changes of the alpha1-beta2 subunit contacts between ligand binding to the alpha and beta subunits of hemoglobin A: UV resonance raman analysis using Ni-Fe hybrid hemoglobin

The alpha1-beta2 subunit contacts in the half-ligated hemoglobin A (Hb A) have been explored with ultraviolet resonance Raman (UVRR) spectroscopy using the Ni-Fe hybrid Hb under various solution conditions. Our previous studies demonstrated that Trpbeta37, Tyralpha42, and Tyralpha140 are mainly responsible for UVRR spectral differences between the complete T (deoxyHb A) and R (COHb A) structures [Nagai, M., Wajcman, H., Lahary, A., Nakatsukasa, T., Nagatomo, S., and Kitagawa, T. (1999) Biochemistry, 38, 1243-1251]. On the basis of it, the UVRR spectra observed for the half-ligated alpha(Ni)beta(CO) and alpha(CO)beta(Ni) at pH 6.7 in the presence of IHP indicated the adoption of the complete T structure similar to alpha(Ni)beta(deoxy) and alpha(deoxy)beta(Ni). The extent of the quaternary structural changes upon ligand binding depends on pH and IHP, but their characters are qualitatively the same. For alpha(Ni)beta(Fe), it is not until pH 8.7 in the absence of IHP that the Tyr bands are changed by ligand binding. The change of Tyr residues is induced by binding of CO, but not of NO, to the alpha heme, while it was similarly induced by binding of CO and NO to the beta heme. The Trp bands are changed toward R-like similarly for alpha(Ni)beta(CO) and alpha(CO)beta(Ni), indicating that the structural changes of Trp residues are scarcely different between CO binding to either the alpha or beta heme. The ligand induced quaternary structural changes of Tyr and Trp residues did not take place in a concerted way and were different between alpha(Ni)beta(CO) and alpha(CO)beta(Ni). These observations directly indicate that the phenomenon occurring at the alpha1-beta2 interface is different between the ligand binding to the alpha and beta hemes and is greatly influenced by IHP. A plausible mechanism of the intersubunit communication upon binding of a ligand to the alpha or beta subunit to the other subunit and its difference between NO and CO as a ligand are discussed.     

The dissociation of NO from nitrosylhemoglobin

The reaction between nitrosylhemoglobin and an excess of deoxymyoglobin has been used to study the kinetics of ligand dissociation from Hb4(NO)4 and Hb4(no)1 species. The kinetics of the dissociation of the first NO molecule from Hb4(no)4 was studied by the ligand replacement method. The results indicate that: (a) the ligand dissociation reaction in Hb4(NO)4 is a cooperative process. This is consistent with the results of Moore and Gibson (Moore, E.G., and Gibson, Q.H. (1977) J. Biol. Chem. 251, 2788-2794). (b) alpha and beta chains in the T state formed by adding IHP to Hb4(NO)4 show kinetic heterogeneity. (c) A similar kinetic heterogeneity is shown by alpha and beta chains in the species Hb4NO in the absence of IHP.(d) The value for the NO dissociation rate constant calculated from the slow phases observed in (b) and (c) is similar to that estimated for the R state. These results suggest that the R to T transition brought about with or without inositol hexaphosphate changes the ligand affinity of one type of the chains much more than of the other. On the basis of IR and EPR studies, it is suggested that alpha chains undergo larger functional changes in R to T transition (or vice versa) in nitrosylhemoglobin. The kinetic parameters for HbNO are compared with those of HbO2 and HbCO and the implications of the results for the reaction mechanism are discussed.     

Evaluation of systemic blood NO dynamics by EPR spectroscopy: HbNO as an endogenous index of NO

The measurement of hemoglobin-nitric oxide (NO) adduct (HbNO) in whole blood by the electron paramagnetic resonance (EPR) method seems relevant for the assessment of systemic NO levels. However, ceruloplasmin and unknown radical species overlap the same magnetic field as that of HbNO. To reveal the EPR spectrum of HbNO, we then introduced the EPR signal subtraction method, which is based on the computer-assisted subtraction of the digitized EPR spectrum of HbNO-depleted blood from that of sample blood using the software. Rats were treated with N(omega)-nitro-L-arginine methyl ester (L-NAME; 120 mg. kg-1. day-1) for 1 wk to obtain HbNO-depleted blood. When this method was applied to the analysis of untreated fresh whole blood, the five-coordinate state of HbNO was observed. HbNO concentration in pentobarbital-anesthetized rats was augmented (change in [HbNO] = 1.6-5.5 microM) by infusion of L-arginine (0.2-0.6 g/kg) but not D-arginine. Using this method, we attempted to evaluate the effects of temocapril on HbNO dynamics in an L-NAME-induced rat endothelial dysfunction model. The oral administration of L-NAME for 2 wk induced a serious hypertension, and the HbNO concentration was reduced (change in [HbNO] = 5.7 microM). Coadministration of temocapril dose dependently improved both changes in blood pressure and the systemic HbNO concentration. In this study, we succeeded in measuring the blood HbNO level as an index of NO by the EPR HbNO signal subtraction method. We also demonstrated that temocapril improves abnormalities of NO dynamics in L-NAME-induced endothelial dysfunction rats using the EPR HbNO signal subtraction method.     

The CO and NO Bohr effect of human hemoglobin with and without inositolhexaphosphate.

Using NO and CO as ligands the Bohr effect of human hemoglobin has been measured with and without inositolhexophosphate. It appears that in the absence and presence of inositolhexaphosphate hemoglobin shows a distinct ligand specificity with respect to the Bohr effect. Ligation with NO is accompanied by release of a larger number of Bohr effect. It is shown that this latter result is due to the fact that the number of protons taken up upon binding of inositolhexaphosphate to ligated hemoglobin is larger for HbNO than for HbCO. It is suggested that this additional proton uptake is partially due to a restoration of the saltbridge between His 146beta and Asp 94beta upon addition of IHP.