Possible mechanism of inhibition of nitrite-induced oxidation of oxyhemoglobin by ergothioneine and uric acid.

The time course of oxyhemoglobin oxidation by nitrite consisted of a kinetic lag followed by a transition phase which progressed into a rapid autocatalytic phase. The imidazolthione and imidazolone derivatives, ergothioneine and uric acid, respectively, caused an increase in the duration of the lag phase in a concentration-dependent manner, without affecting the onset and rate of the autocatalytic phase. Neither compound reacted with H2O2 or nitrite, oxidizing species required in the initiation steps of oxyhemoglobin oxidation. On the other hand, both compounds reduced effectively and at comparable rates the high oxidation state of hemoglobin, i.e., ferrylhemoglobin, which is an intermediate species occurring in the autocatalytic phase. In addition, the rate of ergothioneine oxidation, upon its reaction with ferrylmyoglobin, was accelerated by nitrite, thus suggesting a reaction between the thione and nitrogen dioxide. Nitrogen oxide and ferrylhemoglobin are key species in the free radical chain propagation leading to oxyhemoglobin oxidation by nitrite. These data support the view that ergothioneine and urate delay oxyhemoglobin oxidation by nitrite upon the temporary removal of the propagating species, i.e., nitrogen dioxide and, secondarily, ferrylhemoglobin, and within a mechanism encompassing alterations of the nitrite in equilibrium with nitrogen dioxide and ferrylhemoglobin in equilibrium with methemoglobin redox transitions.     

Mechanism of autocatalytic oxidation of oxyhemoglobin by nitrite. An intermediate detected by electron spin resonance

Oxidation of oxyhemoglobin by nitrite is characterized by the presence of a lag phase followed by the autocatalysis. Just before the autocatalysis begins, an asymmetric ESR signal is detected which is similar to that of the methemoglobin radical generated from methemoglobin and H2O2 in shape, g value (2.005), peak-to-peak width (18 G) and other properties, except the difference in the dependence on temperature. Generation of H2O2 is indicated by the prolongation of the lag phase by the addition of catalase. On the other hand, the oxidation is modified by neither superoxide dismutase nor Nitroblue tetrazolium. The oxidation is prolonged in the presence of KCN. The present results indicate a free-radical mechanism for the oxidation in which the asymmetric radical catalyzes the formation of NO2 from NO2- by a peroxidase action and NO2 oxidizes oxyhemoglobin in the autocatalytic phase.     

Degradation of uric acid during autocatalytic oxidation of oxyhemoglobin induced by sodium nitrite.

The oxidation of oxyhemoglobin produced by sodium nitrite occurs in two stages: 1) an initial slow phase followed by 2) a rapid autocatalytic phase that carries the reaction to completion. The length of the slow phase is extended when uric acid is added to the reaction mixture. As the concentration of uric acid increases, the length of the slow phase increases until a concentration is reached at which the rate of methemoglobin formation is nearly linear until the reaction is complete. Further increases in the concentration of uric acid do not affect the rate of the reaction in the slow phase. At low concentrations of uric acid, where an autocatalytic phase is reached, uric acid is degraded during the reaction. At concentrations of uric acid that keep the reaction in the linear phase, the uric acid is not degraded. It is concluded that uric acid may protect oxyhemoglobin by reacting with HbO2H to yield [HbOH]+ and the urate radical. The urate radical may react with a second molecule of HbO2H and become oxidized. At higher concentrations, the radical may undergo electron transfer with oxyhemoglobin to regenerate the uric acid and form methemoglobin.     

Inhibition by amines indicates involvement of nitrogen dioxide in autocatalytic oxidation of oxyhemoglobin by nitrite

Oxidation of oxyhemoglobin by nitrite is characterized by a lag period followed by an autocatalytic phase. The oxidation can be inhibited by the addition of morpholine, piperidine, triethanolamine or triethylamine (6 mM each). These amines are known to react with nitrogen dioxide to yield nitrosamine. Unexpectedly, aniline or aminopyrine (120 microM each) markedly inhibited the oxidation. These compounds, but not the other amines given above, inhibited the peroxide compound formation from methemoglobin and hydrogen peroxide. The results establish that, during the oxidation, the peroxide compound is generated and converts nitrite into nitrogen dioxide by its peroxidatic activity, resulting in an autocatalytic phase.     

The reaction between nitrite and oxyhemoglobin: a mechanistic study

The nitrite anion (NO(-)(2)) has recently received much attention as an endogenous nitric oxide source that has the potential to be supplemented for therapeutic benefit. One major mechanism of nitrite reduction is the direct reaction between this anion and the ferrous heme group of deoxygenated hemoglobin. However, the reaction of nitrite with oxyhemoglobin (oxyHb) is well established and generates nitrate and methemoglobin (metHb). Several mechanisms have been proposed that involve the intermediacy of protein-free radicals, ferryl heme, nitrogen dioxide (NO(2)), and hydrogen peroxide (H(2)O(2)) in an autocatalytic free radical chain reaction, which could potentially limit the usefulness of nitrite therapy. In this study we show that none of the previously published mechanisms is sufficient to fully explain the kinetics of the reaction of nitrite with oxyHb. Based on experimental data and kinetic simulation, we have modified previous models for this reaction mechanism and show that the new model proposed here is consistent with experimental data. The important feature of this model is that, whereas previously both H(2)O(2) and NO(2) were thought to be integral to both the initiation and propagation steps, H(2)O(2) now only plays a role as an initiator species, and NO(2) only plays a role as an autocatalytic propagatory species. The consequences of uncoupling the roles of H(2)O(2) and NO(2) in the reaction mechanism for the in vivo reactivity of nitrite are discussed.     

Symmetry breaking and the establishment of cell polarity in budding yeast

Cell polarity is typically oriented by external cues such as cell-cell contacts, chemoattractants, or morphogen gradients. In the absence of such cues, however, many cells can spontaneously polarize in a random direction, suggesting the existence of an internal polarity-generating mechanism whose direction can be spatially biased by external cues. Spontaneous 'symmetry-breaking' polarization is likely to involve an autocatalytic process set off by small random fluctuations. Here we review recent work on the nature of the autocatalytic process in budding yeast and on the question of why polarized cells only develop a single 'front'.     

Mechanism of oxyhemoglobin oxidation induced by hydrogen peroxide]

The process of oxyhemoglobin oxidation initiated by hydrogen peroxide in low (10(-7) M) concentrations was investigated. It was found, that H2O2 in this concentration is able to induce the process of chain oxidation of oxyhemoglobin to methemoglobin. The following observations indicate that the process is essentially the chain reaction: 1) The amount of the methemoglobin in haem groups, produced in the reaction, exceed by 20 times the quantity of hydrogen, added initially, to induce the oxidation. 2) Catalase stopped this process at any stage of the reaction. This fact implies that the chain process involves generation of new molecules of H2O2 in the course of oxidation of oxyhemoglobin. The chain reaction proceeded only in the presence of oxygen. But if oxygen was introduced into hemoglobin solution, preincubated with H2O2 in vacuum, than again the oxidation of hemoglobin developed. Apparently, H2O2 in low concentrations appears, mainly, as an inductor of the oxyhemoglobin autooxidation.     

Concerted nitric oxide formation and release from the simultaneous reactions of nitrite with deoxy- and oxyhemoglobin

Recent studies reveal a novel role for hemoglobin as an allosterically regulated nitrite reductase that may mediate nitric oxide (NO)-dependent signaling along the physiological oxygen gradient. Nitrite reacts with deoxyhemoglobin in an allosteric reaction that generates NO and oxidizes deoxyhemoglobin to methemoglobin. NO then reacts at a nearly diffusion-limited rate with deoxyhemoglobin to form iron-nitrosyl-hemoglobin, which to date has been considered a highly stable adduct and, thus, not a source of bioavailable NO. However, under physiological conditions of partial oxygen saturation, nitrite will also react with oxyhemoglobin, and although this complex autocatalytic reaction has been studied for a century, the interaction of the oxy- and deoxy-reactions and the effects on NO disposition have never been explored. We have now characterized the kinetics of hemoglobin oxidation and NO generation at a range of oxygen partial pressures and found that the deoxy-reaction runs in parallel with and partially inhibits the oxy-reaction. In fact, intermediates in the oxy-reaction oxidize the heme iron of iron-nitrosyl-hemoglobin, a product of the deoxy-reaction, which releases NO from the iron-nitrosyl. This oxidative denitrosylation is particularly striking during cycles of hemoglobin deoxygenation and oxygenation in the presence of nitrite. These chemistries may contribute to the oxygen-dependent disposition of nitrite in red cells by limiting oxidative inactivation of nitrite by oxyhemoglobin, promoting nitrite reduction to NO by deoxyhemoglobin, and releasing free NO from iron-nitrosyl-hemoglobin.     

The oxidation of phenylhydrazine: superoxide and mechanism.

The oxidation of phenylhydrazine in buffered aqueous solutions is a complex process involving several intermediates. It can be initiated by metal cations, such as Cu2+; in which case EDTA acts as an inhibitor. It can also be intiated by oxyhemoglobin; in which case chelating agents do not interfere. Superoxide radical is both a product of this reaction and a chain propagator. The formation of O2- could be demonstrated in terms of a reduction of nitroblue tetrazolium, which was prevented by superoxide dismutase. The importance of O2- in carrying the reaction chains was shown by the inhibition of phenylhydrazine oxidation by superoxide dismutase. Hydrogen peroxide accumulated during the reaction and could be detected with catalase. The progress of this oxidation could be monitored in terms of oxygen consumption and by following increases in absorbance at 280 or 320 nm. The oxidation was markedly autocatalytic and superoxide dismutase had the effect of extending the lag period. The absorbance at 280 nm was due to an intermediate which first accumulated and was then consumed. This intermediate appears to be benzendiazonium ion. The absorbance at 320 nm was due to a stable product, which was not identified. The time course of oxygen consumption paralleled the increase in absorbance at 320 nm and lagged behind the changes at 280 nm. Exogenous benzenediazonium ion accelerated the oxidation of phenylhydrazine and eliminated the lag phase. Benzenediazonium ion must therefore react with phenylhydrazine to produce a very reactive intermediate, possibly phenyldiazene. A mechanism was proposed which is consistent with the data. The intermediates and products of the oxidation of phenylhydrazine include superoxide radical, hydrogen peroxide, phenylhydrazyl radical, phenyldiazene, and benzenediazonium ion. This is a minimal list: others remain to be detected and identified. It appears likely that the diverse biological effects of phenylhydrazine are largely due to the reactivities of these intermediates and products.     

The Sugar Model: Autocatalytic Activity of the Triose–Ammonia Reaction

Reaction of triose sugars with ammonia under anaerobic conditions yielded autocatalytic products. The autocatalytic behavior of the products was examined by measuring the effect of the crude triose–ammonia reaction product on the kinetics of a second identical triose–ammonia reaction. The reaction product showed autocatalytic activity by increasing both the rate of disappearance of triose and the rate of formation of pyruvaldehyde, the product of triose dehydration. This synthetic process is considered a reasonable model of origin-of-life chemistry because it uses plausible prebiotic substrates, and resembles modern biosynthesis by employing the energized carbon groups of sugars to drive the synthesis of autocatalytic molecules.     

Kinetic analysis of a simplified scheme of autocatalytic zymogen activation.

Proteolytic enzymes are usually biosynthesized as somewhat larger inactive precursors known as zymogens. These zymogens must undergo an activation process, usually a limited proteolysis, to attain their catalytic activity. When the activating enzyme and the activated enzyme coincide, the process is an autocatalytic zymogen activation. In the present study, a kinetic analysis of the entire progress curve for the autocatalytic zymogen activation reactions is presented. On the basis of the kinetic equations, a novel procedure is developed to evaluate the kinetic parameters of the reactions. This procedure is particularly useful for the fast zymogen autoactivation reactions. As two examples, the novel procedure is used to analyse the autocatalytic activation of bovine trypsinogen and human blood coagulation factor XII (Hageman factor).     

An Auto-Catalytic Surface for Conformational Replication of Amyloid Fibrils—Genesis of an Amyloid World?

Amyloid fibrils are composed of self assembled stacked peptide or protein molecules folded and trapped in a stable cross-beta-sheet conformation. The amyloid fibrillation mechanism represents an intriguing self-catalyzed process rendering replication of a molecular conformational memory of interest for prebiotic chemistry. Herein we describe how a solid surface can be rendered auto-catalytic for fibrillation of a protein solution. We have discovered that a hydrophobic silicon or glass surface can be made to continuously fibrillate solutions of insulin monomers under stressed conditions (pH 1.6, 65°C). It was found that the surface acts as a platform for the formation of nascent seeds that induce fibril replication on and at the surface. This autocatalytic effect stems from a layer a few insulin molecules thick representing an oligomeric layer of misfolded, conformationally trapped, insulin molecules that rapidly through epitaxial growth catalyze the rate determining step (nucleation) during fibril replication. This autocatalytic layer is generated by the protein-solid surface interaction and conformational changes of the adsorbed protein during exposure at the air-water interface. The resulting autocatalytic surface thus both initiates local conformational molecular self-replication and acts as a reservoir for fibril seeds budding off into solution spreading fibril replication entities to the surrounding medium. The possibility of catalysis of the conformational replication process by minute amounts of nucleation sites located on a recruiting surface can evade the issue of dramatic concentration dependence of amyloidogenesis.     

The aggregative stability of human oxyhemoglobin in aqueous media in the presence of mercury(II) compounds]

An analytical review of studies on human oxyhemoglobin coagulation has been performed by the author jointly with V. S. Koniaeva and L. D. Bogdanova within a period from 1985 to 1990. It was shown that the oxyhemoglobin coagulation modified by mercurials proceeded without any essential alteration of native protein conformation. A hypothesis is discussed that the oxyhemoglobin coagulation results from the primary polyaggregation of dimer fragments and that hydrophobic sites which provide for dimer-to-dimer contacts in native tetrameric oxyhemoglobin, participate in this process.     

A density functional theory study of the decomposition mechanism of nitroglycerin

The detailed decomposition mechanism of nitroglycerin (NG) in the gas phase was studied by examining reaction pathways using density functional theory (DFT) and canonical variational transition state theory combined with a small-curvature tunneling correction (CVT/SCT). The mechanism of NG autocatalytic decomposition was investigated at the B3LYP/6-31G(d,p) level of theory. Five possible decomposition pathways involving NG were identified and the rate constants for the pathways at temperatures ranging from 200 to 1000 K were calculated using CVT/SCT. There was found to be a lower energy barrier to the β-H abstraction reaction than to the α-H abstraction reaction during the initial step in the autocatalytic decomposition of NG. The decomposition pathways for CHOCOCHONO₂ (a product obtained following the abstraction of three H atoms from NG by NO₂) include O–NO₂ cleavage or isomer production, meaning that the autocatalytic decomposition of NG has two reaction pathways, both of which are exothermic. The rate constants for these two reaction pathways are greater than the rate constants for the three pathways corresponding to unimolecular NG decomposition. The overall process of NG decomposition can be divided into two stages based on the NO₂ concentration, which affects the decomposition products and reactions. In the first stage, the reaction pathway corresponding to O–NO₂ cleavage is the main pathway, but the rates of the two autocatalytic decomposition pathways increase with increasing NO₂ concentration. However, when a threshold NO₂ concentration is reached, the NG decomposition process enters its second stage, with the two pathways for NG autocatalytic decomposition becoming the main and secondary reaction pathways.     

Enhancement of the autocatalytic activation of trypsinogen to trypsin by bile and bile acids

The activation of trypsinogen to trypsin in the small intestine can occur by the action of enterokinase or, alternatively, as an autocatalytic process catalysed by trypsin itself. We have found that bile salts and human bile cause a significant enhancement of the autocatalytic activation of trypsinogen. This effect is dependent on the calcium ion concentration and is most marked around pH 5.4 and 7.8. An optimum concentration exists for each bile salt at which the greatest enhancement occurs. At this concentration, certain bile salts have been shown to produce activation effects of up to 55-fold. It is suggested that this activation of the autocatalytic process by bile plays an important role in protein digestion in the small intestine, since it has been shown previously that duodenal trypsin levels are abnormally low in patients with an impairment of bile secretion.     

The Divergence and Natural Selection of Autocatalytic Primordial Metabolic Systems

The diversity of the central metabolism of modern organisms is caused by the existence of a few metabolic modules, combination of which produces multiple metabolic pathways. This paper analyzes biomimetically reconstructed coupled autocatalytic cycles as the basis of ancestral metabolic systems. The mechanism for natural selection and evolution in autocatalytic chemical systems may be affected by natural homeostatic parameters such as ambient chemical potentials, temperature, and pressure. Competition between separate parts of an autocatalytic network with positive-plus-negative feedback resulted in the formation of primordial autotrophic, mixotrophic, and heterotrophic metabolic systems. This work examined the last common ancestor of a set of coupled metabolic cycles in a population of protocells. Physical-chemical properties of these cycles determined the main principles of natural selection for the ancestral Bacteria and Archaea taxa.     

Reaction of human hemoglobin with peroxynitrite. Isomerization to nitrate and secondary formation of protein radicals

Peroxynitrite, a strong oxidant formed intravascularly in vivo, can diffuse onto erythrocytes and be largely consumed via a fast reaction (2 x 10(4) m(-1) s(-1)) with oxyhemoglobin. The reaction mechanism of peroxynitrite with oxyhemoglobin that results in the formation of methemoglobin remains to be elucidated. In this work, we studied the reaction under biologically relevant conditions using millimolar oxyhemoglobin concentrations and a stoichiometric excess of oxyhemoglobin over peroxynitrite. The results support a reaction mechanism that involves the net one-electron oxidation of the ferrous heme, isomerization of peroxynitrite to nitrate, and production of superoxide radical and hydrogen peroxide. Homolytic cleavage of peroxynitrite within the heme iron allows the formation of ferrylhemoglobin in approximately 10% yields, which can decay to methemoglobin at the expense of reducing equivalents of the globin moiety. Indeed, spin-trapping studies using 2-methyl-2-nitroso propane and 5,5 dimethyl-1-pyrroline-N-oxide (DMPO) demonstrated the formation of tyrosyl- and cysteinyl-derived radicals. DMPO also inhibited covalently linked dimerization products and led to the formation of DMPO-hemoglobin adducts. Hemoglobin nitration was not observed unless an excess of peroxynitrite over oxyhemoglobin was used, in agreement with a marginal formation of nitrogen dioxide. The results obtained support a role of oxyhemoglobin as a relevant intravascular sink of peroxynitrite.     

Breakage of PrP aggregates is essential for efficient autocatalytic propagation of misfolded prion protein

The conversion of cellular prion protein (PrP(C)) to the disease-associated misfolded isoform (PrP(Sc)) is an essential process for prion replication. This structural conversion can be modelled in protein misfolding cyclic amplification (PMCA) reactions in which PrP(Sc) is inoculated into healthy hamster brain homogenate, followed by cycles of incubation and sonication. In serial transmission PMCA experiments it has recently been shown that the protease-resistant PrP obtained in vitro (PrPres) is generated by an autocatalytic mechanism. Here, serial transmission PMCA experiments were compared with serial transmission reactions lacking the sonication steps. We achieved approximately 200,000-fold PrPres amplification by PMCA. In contrast, although initial amplification was comparable to PMCA reactions, PrPres levels quickly dropped below detection limit when samples were not subjected to ultrasound. These results indicate that aggregate breakage is essential for efficient autocatalytic amplification of misfolded prion protein and suggest an important role of aggregate breakage in prion propagation.     

Autocatalytic cooperativity and self-regulation of ATPase pumps in membrane active transport

We investigate the effect of autocatalysis on the conformational changes of membrane pumps during active transport driven by ATP. The translocation process is described by means of an alternating access model. The usual kinetic scheme is extended by introducing autocatalytic steps and allowing for dynamic formation of enzyme complexes. The usual features of cooperative models are recovered, i.e., sigmoid shapes of flux versus concentration curves. We show also that two autocatalytic steps lead to a mechanism of inhibition by the substrate as experimentally observed for some ATPase pumps. In addition, when the formation of enzyme complexes is allowed, the model exhibits a multiple stationary states regime, which can be related to a self-regulation mechanism of the active transport in biological systems. Correspondence to: G. Weissmüller     

The demonstration of ferrihemochrome intermediates in heinz body formation following the reduction of oxyhemoglobin A by acetylphenylhydrazone.

Interaction of acetylphenylhydrazine with oxyhemoglobin A in a hemolysate or in intact red cells resulted in the formation of ferrihemochromes as shown by a characteristic optical spectrum. The same optical spectrum was observed in a suspension of red cell ghosts containing numerous Heinz bodies. Electron paramagnetic resonance of actylphenylhydrazine-incubated red cells disclosed the presence of previously identified reversible ferrihemochromes, which can be reduced to functional hemoglobin, and irreversible ferrihemochromes, which cannot be reduced to functional hemoglobin. (Ferrihemochromes are defined as low spin forms of ferric hemoglobin having heme ligands endogenous to the protein structure). In contrast, only irreversible ferrihemochromes could be observed in ghosts containing Heinz bodies. In addition both optical and magnetic features of sulfhemoglobin were observed in an acetylphenylhydrazine-treated red cell hemolysate. Similar optical features are produced by the interaction of aromatic nitrogen-containg reductants with purified oxyhemoglobin in the presence of (NH4)2S. This reaction is not effected by the presence of catalase, suggesting that H2O2 is not an intermediate of the reaction. It is concluded that the mechanism of action of acetylphenylhydrazine with oxyhemoglobin is two-fold, ultimate reduction to high spin ferric hemoglobin followed by ferrihemochrome formation. Thus it appears that the pathway of denaturation of hemolytic anemias and thalassemia or induced by chemical reagents, entails a common route involving the formation of ferric hemoglobin by a reductive mechanism, followed by reversible ferrihemochromes, irreversible ferrihemochromes, and ultimately, precipitation.