Kinetic study of catalytic CO(2) hydration by water-soluble model compound of carbonic anhydrase and anion inhibition effect on CO(2) hydration

A kinetic study of CO(2) hydration was carried out using the water-soluble zinc model complex with water-soluble nitrilotris(2-benzimidazolylmethyl-6-sulfonate) L1S, [L1SZn(OH(2))](-), mimicking the active site of carbonic anhydrase, in the presence and absence of anion inhibitors NCS(-) and Cl(-). The obtained rate constants k(cat) for CO(2) hydration were 5.9x10(2), 1. 7x10(3), and 3.1x10(3) M(-1) s(-1) at 5, 10, and 15 degrees C, respectively: the k(cat)=ca. 10(4) M(-1) s(-1) extrapolated towards 25 degrees C has been the largest among the reported k(cat) using zinc model complexes for carbonic anhydrase. It was also revealed that NCS(-), Cl(-) and acetazolamide play a role of inhibitors by the decrease of k(cat): 7x10(2) and 2x10(3) M(-1) s(-1) for NCS(-) and Cl(-) at 15 degrees C, respectively. The sequence of their magnitudes in k(cat) is Cl(-) approximately acetazolamide>NCS(-), where the sequence Cl(-)>NCS(-) is confirmed for native carbonic anhydrase. The difference of k(cat) or k(obs) between NCS(-) and Cl(-) resulted from that between the stability constants K(st)=2x10(3) for [L1SZn(NCS)](2-) and 1x10(2) M(-1) for [L1SZnCl](2-) in D(2)O: for water-insoluble tris(2-benzimidazolylmethyl)amine L1, K(st)=1.8x10(4) for [L1Zn(NCS)](2-) and 1.5x10(3) M(-1) for [L1ZnCl](2-)in CD(3)CN/D(2)O (50% v/v). The crystal structure of anion-binding zinc model complexes [L1Zn(OH(2))](0.5)[L1ZnCl](0.5) (ClO(4))(1.5) 1(0.5)2(0.5)(ClO(4))(1.5) was revealed by X-ray crystallography. The geometry around Zn(2+) in 1 and 2 was tetrahedrally coordinated by three benzimidazolyl nitrogen atoms and one oxygen atom of H(2)O, or Cl(-).     

Ca2+ binding effects on the C2 domain conformation of human cytosolic phospholipase A2

It has been reported that the cooperative binding of calcium ions indicated a local conformational change of the human cytosolic phospholipase A2 (cPLA2) C2 domain (Nalefski et al., (1997) Biochemistry 36, 12011-12018). However its structural evidence is less known (Malmberg et al., (2003) Biochemistry 42, 13227-13240). In this letter, life-time decay and fluorescence quenching techniques were employed to compare the calcium-induced conformational changes. The life-time decay parameters and fluorescence quenching constant changes were small between the apo- and holo-C2 domains when tryptophan residue was excited at 295 nm. In contrast, the quenching constant change was large, from 0.52 M(-1) for the apo-C2 to 8.8 M(-1) for the holo-C2 domain, when tyrosine residues were excited at 284 nm. Our results provide new information on amino acid side chain orientation change at calcium binding loop 3, which is necessary for Ca2+ binding regulated membrane targeting of human cytosolic phospholipase A2.     

Raman signatures of ligand binding and allosteric conformation change in hexameric insulin.

Hexameric insulin is an allosteric protein that undergoes transitions between three conformational states (T(6), T(3)R(3), and R(6)). These allosteric states are stabilized by the binding of ligands to the phenolic pockets and by the coordination of anions to the His B10 metal sites. Raman difference (RD) spectroscopy is utilized to examine the binding of phenolic ligands and the binding of thiocyanate, p-aminobenzoic acid (PABA), or 4-hydroxy-3-nitrobenzoic acid (4H3N) to the allosteric sites of T(3)R(3) and R(6). The RD spectroscopic studies show changes in the amide I and III bands for the transition of residues B1-B8 from a meandering coil to an alpha helix in the T-R transitions and identify the Raman signatures of the structural differences among the T(6), T(3)R(3), and R(6) states. Evidence of the altered environment caused by the approximately 30 A displacement of phenylalanine (Phe) B1 is clearly seen from changes in the Raman bands of the Phe ring. Raman signatures arising from the coordination of PABA or 4H3N to the histidine (His) B10 Zn(II) sites show these carboxylates give distorted, asymmetric coordination to Zn(II). The RD spectra also reveal the importance of the position and the type of substituents for designing aromatic carboxylates with high affinity for the His B10 metal site.     

The multifunctional oxidase activity of ceruloplasmin as revealed by anion binding studies.

The effect of multiple binding of azide, N3-, on the structural and functional properties of ceruloplasmin (CP) has been reinvestigated by means of both spectroscopic and enzymatic techniques. High affinity binding of the anion to human CP resulted in a dramatic increase of the absorbance at 610 nm and in a concomitant decrease of the optical density at 330 nm. The oxidase activity toward Fe(II) was essentially unaffected, while turnover parameters versus nonferrous substrates dramatically changed, with an approximately 100-fold enhancement of the kcat/Km parameter. Chloride at physiological concentration proved to behave very similarly to N3- bound with high affinity, in that it not only induced the spectroscopic changes previously interpreted in terms of an intramolecular electron transfer from reduced type 1 to type 3 copper ions [Musci, G., Bonaccorsi di Patti, M.C. & Calabrese, L. (1995) J. Protein Chem. 14, 611-617], but it also enhanced some 60-fold the kcat/Km value. A different behavior was observed with chicken CP, where a decrease at 330 nm occurred without a concomitant modification at 603 nm. The chicken enzyme was less sensitive also in terms of enzymatic activity, which was nearly unchanged in the presence of either high affinity N3- or Cl-. At higher N3- concentrations, optical changes of both human and chicken CP were mainly focussed on the appearance of ligand-to-metal charge transfer bands below 500 nm, and the anion behaved as an inhibitor of the oxidase activity versus Fe(II) as well as noniron substrates. The well known bleaching of the blue chromophore could be observed, at neutral pH, only at very high N3-/CP ratios. The data presented in this paper are consistent with a mechanism of structural and functional modulation of CP by anions, that would be able to dictate the substrate specificity of the cuproprotein, and suggest the possibility that CP may act in vivo as a multifunctional oxidase.     

Low-Temperature conformation of Mg2+-Poly(U) in D2O as revealed by IR and Raman Spectroscopy and by normal-mode analysis treatment

Infrared and Raman spectra of the Mg²⁺ salt of poly(U) in D₂O were recorded in the 1600-1800 cm^?1 region and between 1 and 20C. The ir spectra showed a melting curve similar to the uv melting curves with a temperature of transition of about 6.5°C. This spectral change is assumed to be associated with the formation of the secondary structure of Mg²⁺-poly(U) in D₂O at this temperature. Three double-helical and two triple-helical structures were used as inputs to compute the normal modes of vibration. A double-helical structure was found to give the best agreement with the observations. Knowledge of the C=0 eigenvectors, and of the expression for transition probability from quantum mechanics, was used to explain the so far unanswered question of H. T. Miles [(1964) Proc. Natl. Acad. Sci. USA 51, 1104–1109; (1980) Biomolecular Structure, Conformation, Function and Evolution, Pergamon, Oxford, pp. 251–264] as to why there is an increase in the ir vibrational wave number of a carbonyl band when that group is H-bonded to another polynucleotide chain in a helix. Such considerations also explain why a predicted band at about 1648 cm^?1 is not to be seen in the ir spectra but is present in the Raman spectra. The model incorporating the C?O transition dipole-dipole coupling interaction is able to explain also the observed higher intensity of the higher wave-number ir band. The experimental results demonstrate that the complete picture of vibrational dynamics of Mg²⁺-poly(U) in D₂O is obtained only by looking simultaneously at ir and Raman spectra and not at only one of them. Weak ir bands were found to be as useful as the strong ones in understanding structure and vibrational dynamics. On the bases of our ir and Raman spectra, of the normal-mode analyses, and of the literature data, it is concluded that Mg²⁺-poly(U) in D₂O is present in a double-helical structure at temperatures below the temperature of transition, whereby the uracil residues are paired according to arrangement (a) (see Fig. 1). This structure is rodlike and arises by refolding of one poly(U) chain. The computations show that no normal mode is associated with a single C?O group vibration; all C?O group vibrations are heavily mixed motions of various C?O groups.     

Ligand migration in human myoglobin: steric effects of isoleucine 107(G8) on O(2) and CO binding

To investigate the ligand pathway in myoglobin, some mutant myoglobins, in which one of the amino acid residues constituting a putative ligand-docking site, Ile107, is replaced by Ala, Val, Leu, or Phe, were prepared and their structural and ligand binding properties were characterized. The kinetic barrier for the ligand entry to protein inside was lowered by decreasing the side-chain volume at position 107, indicating that the bulky side chain interferes with the formation of the activation state for the ligand migration and the free space near position 107 would be filled with the ligand in the activation state. Another prominent effect of the reduced side-chain volume at position 107 is to stabilize the ligand-binding intermediate state. Because the stabilization can be ascribed to decrease of the positive enthalpy, the enlarged free space near position 107 would relieve unfavorable steric interactions between the ligand and nearby amino acid residues. The side-chain volume at position 107, therefore, is crucial for the kinetic barrier for the ligand migration and free energy of the ligand-binding intermediate state, which allows us to propose that some photodissociated O(2) moves toward position 107 to be trapped and then expelled to the solvent.     

Novel allosteric effectors of the tryptophan synthase alpha(2)beta(2) complex identified by computer-assisted molecular modeling

Tryptophan synthase is a pyridoxal 5'-phosphate-dependent alpha(2)beta(2) complex catalyzing the formation of L-tryptophan. The functional properties of one subunit are allosterically regulated by ligands of the other subunit. Molecules tailored for binding to the alpha-active site were designed using as a starting model the three-dimensional structure of the complex between the enzyme from Salmonella typhimurium and the substrate analog indole-3-propanol phosphate. On the basis of molecular dynamics simulations, indole-3-acetyl-X, where X is glycine, alanine, valine and aspartate, and a few other structurally related compounds were found to be good candidates for ligands of the alpha-subunit. The binding of the designed compounds to the alpha-active site was evaluated by measuring the inhibition of the alpha-reaction of the enzyme from Salmonella typhimurium. The inhibition constants were found to vary between 0.3 and 1.7 mM. These alpha-subunit ligands do not bind to the beta-subunit, as indicated by the absence of effects on the rate of the beta-reaction in the isolated beta(2) dimer. A small inhibitory effect on the activity of the alpha(2)beta(2) complex was caused by indole-3-acetyl-glycine and indole-3-acetyl-aspartate whereas a small stimulatory effect was caused by indole-3-acetamide. Furthermore, indole-3-acetyl-glycine, indole-3-acetyl-aspartate and indole-3-acetamide perturb the equilibrium of the catalytic intermediates formed at the beta-active site, stabilizing the alpha-aminoacrylate Schiff base. These results indicate that (i) indole-3-acetyl-glycine, indole-3-acetyl-aspartate and indole-3-acetamide bind to the alpha-subunit and act as allosteric effectors whereas indole-3-acetyl-valine and indole-3-acetyl-alanine only bind to the alpha-subunit, and (ii) the terminal phosphate present in the already known allosteric effectors of tryptophan synthase is not strictly required for the transmission of regulatory signals.     

Effect of methylamine and plasmin on the conformation of human alpha 2-macroglobulin as revealed by differential scanning calorimetric analysis.

Differential scanning calorimetric analysis was used as a probe of the conformational alteration in human alpha 2-macroglobulin (AM) upon its complex formation with methylamine and with the protease, human plasmin. The slow electrophoretic form of AM displayed a single thermal transition, characterized by a temperature midpoint (Tm) of 65.8 +/- 0.3 degrees, a calorimetric enthalpy (delta Hc) of 2,550 +/- 150 kcal/mol and a van't Hoff enthalpy (delta Hvh) of 140 kcal/mol. In the presence of sufficient methylamine to irreversibly disrupt the four thiol ester bonds in AM, a single thermal transition was obtained, characterized by a Tm of 62.8 +/- 0.3 degrees, a delta Hc of 1,700 +/- 100 kcal/mol, and a delta Hvh of 169 kcal/mol. These data suggest that a major conformational alteration is produced in AM upon complex formation with methylamine. When plasmin interacts with AM, the resulting thermogram displays Tm values for AM of 68-69 degrees and 77 degrees, also suggestive of a large conformational alteration in AM. However, this latter alteration appears dissimilar to the change induced by methylamine.     

Novel allosteric sites on human erythrocyte acetylcholinesterase identified by two monoclonal antibodies.

Monoclonal antibodies against human erythrocyte acetylcholinesterase (acetylcholine acetylhydrolase EC 3.1.1.7) have been examined for inhibition of enzyme activity. Of sixteen antibodies analyzed, only one (C1B7) inhibited enzyme activity, indicating selection of an unusual susceptible site. The inhibitory activity of C1B7 was characterized and compared to another inhibitory antibody, AE-2, previously described by Fambrough et al. (Proc. Natl. Acad. Sci. USA 79, 1078, 1982). Maximal demonstrated inhibition was 84% for C1B7 and 72% for AE-2 and antibody inhibition of enzyme activity was equivalent for the reduced and alkylated acetylcholinesterase monomer and the intact dimer. The Ki (stoichiometry of the enzyme-antibody reaction estimated from enzyme kinetics) was 1.0 for C1B7 and 4.8 molecules of antibody per monomer of acetylcholinesterase for AE-2. The antibodies did not compete with one another for binding to acetylcholinesterase, indicating that they have different target epitopes on the enzyme. Antibody binding to the enzyme was not specifically affected by any of the anticholinesterase agents tested: (a) the irreversible esteratic site-directed inhibitor diisopropylfluorophosphate; (b) the reversible active site-directed inhibitors edrophonium, neostigmine, BW284c51, and carbachol; and (c) allosteric site-directed compounds propidium and gallamine. Kinetic analysis of their effects provide evidence that both antibodies decrease the catalytic rate of enzyme activity and have little or no effect on substrate binding.     

The Na+ binding channel of human coagulation proteases: novel insights on the structure and allosteric modulation revealed by molecular surface analysis

Thrombovascular diseases result from imbalanced haemostasis and comprise important health problems in the aging population worldwide. The activity of enzymes pertaining to the coagulation cascade of mammalians exhibit several control mechanisms in order to maintain a proper balance between bleeding and thrombosis. For instance, human coagulation serine proteases carrying a F225 or Y225 are allosteric modulated by the binding of Na+ in a water-filled channel connected to the primary specificity pocket (S1 subsite) of these enzymes. We have characterized the structure, topography and lipophilicity of this channel in the ligand-free fast (sodium-bound) and slow (sodium-free) forms of thrombin, in the sole available structure of activated protein C and in several structures of the coagulation factors VIIa, IXa and Xa, differing in the nature of the bound inhibitor and in the occupancy of exosite-I as well as the Ca2+ and Na+ binding sites. Opposite to thrombin, the aqueous channels in all other coagulation enzymes sheltering a Na+ binding site do not have an aperture on the enzyme surface opposite to the S1 subsite entrance. In these enzymes, the lack of the three-residue insertion in loop 1 (183-189) as found in thrombin allied to compensatory mutations in the positions 187-185 and 222 effects a constriction in the water-filled channel that ends up by segregating the ion binding site from the S1 subsite. We also disclosed major topographical changes on the thrombin's surface upon sodium release and transition to the slow form that culminate in the narrowing of the S1 subsite entrance and, strikingly, in the loss of communication between the primary specificity pocket and the exosite-I. Such observation is in accordance with existing experimental data demonstrating thermodynamic linkage between these distant regions on the thrombin surface. Conformational changes in F34, L40, R73 and T74 were the main responsible for this effect. A path by which these changes in the vicinity of exosite-I could be transmitted to the S1 subsite and, consequently, to the sodium binding site is proposed.     

Kinetics of CO and O(2) binding to human serum albumin-heme hybrid

The kinetics of the CO and O(2) binding to the synthetic hemoprotein, recombinant human serum albumin (rHSA) incorporating eight 2-[8-?N-(2-methylimidazolyl)?octanoyloxymethyl]-5,10,15, 20-tetrakis(o-pivalamido)phenylporphinatoiron(II)s (FePs) [rHSA-FeP(8)] have been investigated by laser flash photolysis. Time dependence of the absorption change accompanied the CO rebinding to rHSA-FeP(8) was composed of three phases. The fastest component was the axial base elimination, and the long-lived biphasic decay corresponds to the direct recombination of CO to the five-N-coordinated FePs in rHSA. The rate constants of the fast and slow phases of the CO association [(fast), (slow)] were determined to be 4.9 x 10(6) M(-)(1) s(-)(1) and 6.7 x 10(5) M(-)(1) s(-)(1), respectively. The initial amplitude after the laser pulse gave the concentration ratio of the fast and slow phases (n = 3); (i) two of the eight FePs exhibited the slow rate constants and (ii) they are presumably accommodated in the second and fifth binding sites of FeP in the albumin structure. The absorption decay following the O(2) photodissociation of rHSA-FeP(8) also showed the same behavior. Thermodynamically, the large DeltaG() of the slow phase of the CO rebinding, which mainly comes from the enthalpic factor, suggests the appearance of additional steric hindrance on the central metal iron of FeP. Furthermore, orientation of the porphyrin plane in rHSA was predicted by molecular simulation, which supports the experimental data from the kinetic observations.     

A study into the effects of protein binding on nucleotide conformation.

In this study, we examine the effects of binding to protein upon nucleotide conformation, by the comparison of X-ray crystal structures of free and protein-bound nucleotides. A dataset of structurally non-homologous protein-nucleotide complexes was derived from the Brookhaven Protein Data Bank by a novel protocol of dual sequential and structural alignments, and a dataset of native nucleotide structures was obtained from the Cambridge Structural Database. The nucleotide torsion angles and sugar puckers, which describe nucleotide conformation, were analysed in both datasets and compared. Differences between them are described and discussed. Overall, the nucleotides were found to bind in low energy conformations, not significantly different from their 'free' conformations except that they adopted an extended conformation in preference to the 'closed' structure predominantly observed by free nucleotide. The archetypal conformation of a protein-bound nucleotide is derived from these observations.     

Crowding and hydration effects on protein conformation: a study with sol-gel encapsulated proteins.

We are developing an experimental system for testing the effects of macromolecular crowding and molecular confinement on protein structure. In the present study, solvent effects on the secondary structure of two proteins were examined by circular dichroism following encapsulation in the hydrated pores of a silica glass matrix by the sol-gel method. Changes in the unfolded conformations of encapsulated apomyoglobin and reduced serum albumin were analyzed after equilibration with aqueous solutions of natural osmolytes, short-chain alcohols, polyethylene glycol, and a complete series of Hofmeister cations. In many instances, the alpha-helical content of the encapsulated protein was increased by addition of solutes at concentrations that have no effect on the protein in the absence of the glass. The results are discussed from the perspective of water structure. We argue that perturbed water at the silica interface causes an increase in the average free energy of the bulk water phase which, consequently, diminishes the strength of the hydrophobic effect inside the glass matrix and destabilizes the conformation of encapsulated proteins. We propose that solutes can increase the strength of the hydrophobic effect and influence folding equilibria without directly interacting with the protein. A hypothesis is provided for the apparent paradox that kosmotropic (strongly water binding) anions favor native protein structure, whereas chaotropic (weakly water binding) cations enhance native protein structure. The encapsulation results suggest that macromolecular crowding and molecular confinement are accompanied by hydration effects that may oppose or potentiate the stabilizing effects of excluded volume on protein structure, depending on the surface chemistry of the crowding agent and its influence on bulk water structure. In the crowded environment of a living cell, excluded volume effects, surface-induced water structure, and compatible solutes are expected to complement the dominant forces in protein folding.     

Effects of the transport site conformation on the binding of external NAP-taurine to the human erythrocyte anion exchange system. Evidence for intrinsic asymmetry

External N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine) inhibits human red cell chloride exchange by binding to a site that is distinct from the chloride transport site. Increases in the intracellular chloride concentration (at constant external chloride) cause an increase in the inhibitory potency of external NAP-taurine. This effect is not due to the changes in pH or membrane potential that usually accompany a chloride gradient, since even when these changes are reversed or eliminated the inhibitory potency remains high. According to the ping-pong model for anion exchange, such transmembrane effects of intracellular chloride on external NAP-taurine can be explained if NAP-taurine only binds to its site when the transport site is in the outward-facing (Eo or EClo ) form. Since NAP-taurine prevents the conformational change from EClo to ECli , it must lock the system in the outward-facing form. NAP-taurine can therefore be used just like the competitive inhibitor H2DIDS (4,4'-diisothiocyano-1,2- diphenylethane -2,2'-disulfonic acid) to monitor the fraction of transport sites that face outward. A quantitative analysis of the effects of chloride gradients on the inhibitory potency of NAP-taurine and H2DIDS reveals that the transport system is intrinsically asymmetric, such that when Cli = Clo, most of the unloaded transport sites face the cytoplasmic side of the membrane.     

Effects of polyvalent anion binding to hemoglobin on oxygen and oxidation-reduction equilibria and their relevance to allosteric transition.

The reaction of human hemoglobin and some of its derivatives with aliphatic and aromatic compounds carrying from two to six carboxylate groups has been studied. The effect of the polycarboxylates as well as of three co-ordinate anions (respectively tri-, tetra- and pentavalent) on the oxygenation and oxidation-reduction equilibria and optical spectra have been compared to those of 2,3-diphosphoglycerate and inositol hexaphosphate.All the polyvalent anions raise the P_0.5³ and the E_m values of human hemoglobin and are thus bound more strongly to deoxyhemoglobin than to oxy- or methemoglobin. Binding of benzene hexacarboxylate and benzene pentacarboxylate to oxyhemoglobin is demonstrated through a study of oxygenation curves, that of these reagents, and ferrocyanide, to methemoglobin through their effect on redox potential as well as on optical spectra. Methemoglobin and oxyhemoglobin are shown to bind more than one molecule of the carboxylates at high anion concentrations. Results bearing on the anion binding site for deoxy- as well as for methemoglobin are reported.Two appropriate human hemoglobin derivatives, namely Hb_BME and Hb_{NES desArg} have been examined in search of relations between the effect of anions on oxygen equilibria and that on quaternary structure: in both of these derivatives the chemical modifications inhibit quaternary conformational change that would result from oxygen binding, the deoxy structure being strongly destabilized. Several of the polyanions significantly raise the P_0.5 values of these derivatives but do not modify the quaternary structure, as judged from the absence of characteristic spectral changes. The results imply that anion binding by these proteins somehow inhibits the change in tertiary structure produced by oxygen binding; similar considerations may also apply in the case of the normal hemoglobin-diphosphoglycerate complex.     

Acute vs. chronic effects of elevated hemoglobin O(2) affinity on O(2) transport in maximal exercise.

These studies were conducted to compare the effects on systemic O(2) transport of chronically vs. acutely increased Hb O(2) affinity. O(2) transport during maximal normoxic and hypoxic [inspired PO(2) (PI(O(2))) = 70 and 55 Torr, respectively] exercise was studied in rats with Hb O(2) affinity that was increased chronically by sodium cyanate (group 1) or acutely by transfusion with blood obtained from cyanate-treated rats (group 2). Group 3 consisted of normal rats. Hb O(2) half-saturation pressure (P(50); Torr) during maximal exercise was approximately 26 in groups 1 and 2 and approximately 46 in group 3. In normoxia, maximal blood O(2) convection (TO(2 max) = cardiac output x arterial blood O(2) content) was similar in all groups, whereas in hypoxia TO(2 max) was significantly higher in groups 1 and 2 than in group 3. Tissue O(2) extraction (arteriovenous O(2) content/arterial O(2) content) was lowest in group 1, intermediate in group 2, and highest in group 3 (P < 0.05) at all exercise PI(O(2)) values. In normoxia, maximal O(2) utilization (VO(2 max)) paralleled O(2) extraction ratio and was lowest in group 1, intermediate in group 2, and highest in group 3 (P < 0.05). In hypoxia, the lower O(2) extraction ratio values of groups 1 and 2 were offset by their higher TO(2 max); accordingly, their differences in VO(2 max) from group 3 were attenuated or reversed. Tissue O(2) transfer capacity (VO(2 max)/mixed venous PO(2)) was lowest in group 1 and comparable in groups 2 and 3. We conclude that lowering Hb P(50) has opposing effects on TO(2 max) and O(2) extraction ratio, with the relative magnitude of these changes, which varies with PI(O(2)), determining VO(2 max). Although the lower O(2) extraction ratio of groups 2 vs. 3 suggests a decrease in tissue PO(2) diffusion gradient secondary to the low P(50), the lower O(2) extraction ratio of groups 1 vs. 2 suggests additional negative effects of sodium cyanate and/or chronically low Hb P(50) on tissue O(2) transfer.     

Inhibitory and enhancing effects of NO on H(2)O(2) toxicity: dependence on the concentrations of NO and H(2)O(2)

Nitric oxide (NO) has been shown to both enhance hydrogen peroxide (H(2)O(2)) toxicity and protect cells against H(2)O(2) toxicity. In order to resolve this apparent contradiction, we here studied the effects of NO on H(2)O(2) toxicity in cultured liver endothelial cells over a wide range of NO and H(2)O(2) concentrations. NO was generated by spermine NONOate (SpNO, 0.001-1 mM), H(2)O(2) was generated continuously by glucose/glucose oxidase (GOD, 20-300 U/l), or added as a bolus (200 microM). SpNO concentrations between 0.01 and 0.1 mM provided protection against H(2)O(2)-induced cell death. SpNO concentrations >0.1 mM were injurious with low H(2)O(2) concentrations, but protective at high H(2)O(2) concentrations. Protection appeared to be mainly due to inhibition of lipid peroxidation, for which SpNO concentrations as low as 0.01 mM were sufficient. SpNO in high concentration (1 mM) consistently raised H(2)O(2) steady-state levels in line with inhibition of H(2)O(2) degradation. Thus, the overall effect of NO on H(2)O(2) toxicity can be switched within the same cellular model, with protection being predominant at low NO and high H(2)O(2) levels and enhancement being predominant with high NO and low H(2)O(2) levels.