Preparation of thrombokinase from bovine plasma

Thrombokinase is prepared from bovine plasma by a procedure involving: treatment with diatomaceous silica, adsorption on barium sulfate, flowing elution with two successive phosphate buffers, ammonium sulfate fractionation, "spontaneous" activation in concentrated solution, and isoelectric precipitation. The yield of nitrogen is 0.002 per cent, corresponding to 1.2 mg. protein per liter of plasma. When diluted back to the volume of parent plasma, and complemented by calcium plus cephalin, the product causes appreciable activation of prothrombin in 1 minute. Thus, the quantity of thrombokinase obtainable is compatible with a physiologic role. In the more complex system used for routine assay, thrombokinase can be supplied by crude plasma at a dilution of 1/500. In parallel tests, the product appears to be more active than its parent plasma, although it contains only 0.002 per cent of the nitrogen. However, the thrombokinase of the product has been activated, whereas the thrombokinase of the plasma is probably in an inactive precursor state. When diluted back to the volume of parent plasma, to a concentration of 0.2 microgram nitrogen per ml., thrombokinase can slowly activate prothrombin in the presence of oxalate, and without the addition of accessory factors. Activation of prothrombin in the presence of oxalate is faster with higher concentrations of thrombokinase.     

Effect of blood thrombokinase,as influenced by soy bean trypsin inhibitor,ultracentrifugation, and accessory factors

1. Crystallized soy bean trypsin inhibitor, at a concentration of 100 microg./ml., suppressed the production of thrombin from a mixture of prothrombin and blood thrombokinase. The experiment was performed in the presence of 0.011 M oxalate, in order to minimize the possibility of participation by accessory factors which require ionic calcium. The results are in accord with the view that thrombokinase is a trypsin-like enzyme. 2. When a solution of blood thrombokinase was centrifuged at 85,000 g for 120 minutes, almost all the activity remained in the supernate. This supernate activated the supernate from a prothrombin solution which had been similarly centrifuged. The activation of prothrombin by thrombokinase can proceed in the absence of material completely sedimentable in 120 minutes at 85,000 g. 3. An "accelerator" reagent was prepared by treating bovine serum with barium carbonate, and then passing the serum through a column of diatomaceous earth. This "accelerator" was used together with prothrombin, blood thrombokinase, Howell's cephalin, and calcium chloride to compose a five-reagent thrombin-producing system. In this system, no thrombin was produced without thrombokinase. On the other hand, thrombin was produced from prothrombin and thrombokinase, even when all the other reagents were omitted. When calcium was omitted, thrombokinase was able to function; but cephalin and the "accelerator" reagent were ineffective. 4. Quantitative tests indicated that the "accelerator" reagent exerted an effect distinct from those of thrombokinase and cephalin. However, it is not certain whether the "accelerator" reagent functioned as an accessory factor, as a potential source of more thrombokinase, or both. In the experiments reported, thrombokinase was primary to, or necessary for, the effect of "accelerator." 5. The effectiveness of thrombokinase was multiplied a hundred times or more, when complemented by calcium, cephalin, and "accelerator" reagent. Ionic calcium was a necessary component of this complementing system. This may help to explain why removal of calcium ions keeps blood fluid, even though thrombokinase, by itself, is little influenced either by calcium ions or by oxalate.     

Outstanding Characteristics of Thrombokinase Isolated from Bovine Plasma

Thrombokinase has been isolated from bovine plasma by a procedure which begins with the highly purified product of a previously described method, chromatographs it on DEAE-cellulose, and then fractionates it by continuous flow electrophoresis, yielding 0.2 mg per liter of oxalated plasma. The electrophoretic fraction has shown a single boundary in the ultracentrifuge; and its esterase activity on toluenesulfonylarginine methyl ester has been about the same as that of thrombokinase previously isolated by repeated electrophoretic fractionations. Thrombokinase is a euglobulin with minimum solubility near pH 5.0. It is most stable within the pH range 7.5 to 9.5; but there is also a peak in the stability curve near pH 1.8. A few micrograms of thrombokinase per milliliter can activate prothrombin in the presence of EDTA. A few thousandths of a microgram causes rapid production of thrombin in the system: prothrombin, thrombokinase, calcium chloride, phosphatide, "accelerator." But, thrombokinase has less than 1/175 the proteolytic activity of crystallized trypsin.     

Thrombokinase of the Blood as Trypsin-Like Enzyme

Thrombokinase of the blood, while resembling enterokinase in its role of activator, is more closely analogous to trypsin in its intrinsic origin. It probably arises from a plasma precursor; but it is different from plasmin (fibrinolysin). Like trypsin, thrombokinase can activate prothrombin without the aid of other factors; however, it is potentiated by platelets plus calcium. Unlike certain tissue "thromboplastins," it does not sediment appreciably in 2 hours at 85,000 g. Like trypsin, it hydrolyzes p-toluenesulfonylarginine methyl ester (TAMe). Chromatography on DEAE-cellulose separated thrombin from thrombokinase. The TAMe esterase associated with the thrombokinase fractions was largely suppressed by soybean trypsin inhibitor, while that associated with the thrombin fractions was not. Highly purified thrombokinase was used as starting material; and thrombokinase was eluted in the last major protein band. Under these conditions stepwise elution was as effective as gradient in leading to further purification. The product of 199 liters of bovine plasma was chromatographed in 1 day; and the specific activity was comparable to that attained previously by repeated electrophoretic fractionations. The assembled data suggest that the thrombokinase protein may be approaching homogeneity.     

A note on the mathematical theory of the effect of nervous stress on coronary thrombosis

The “classical” view of formation of blood clots is that the disintegration of platelets releases thrombokinase which activates prothrombin into thrombin. The latter combining with fibrinogen produces fibrin, the substance of the clot. There is, however, ample evidence of hormonal and other biochemical factors as playing a role in the process of clotting. Through the necessity of a breakdown of platelets has been challenged, yet theirpresence is considered as necessary for clotting. Thefirst part of this paper is based on the assumption that either a breakdown or even an injury of a platelet produces thrombokinase. An injury is likely to occur due to collisions between platelets, between platelets and erythrocytes or between platelets and leukocytes. A probability is ascribed to the production of thrombokinase by a collision. Thus not every collision is effective. The frequency of those collisions is calculated according to classical physics. Inasmuch as mental stress usually causes vasoconstriction and therefore an increase of concentration of the formed elements of blood, the frequency of collisions and therefore the production of small amounts of thrombokinase increases with stress. If thrombokinase formed by the collision remains preserved and therefore accumulates in the blood, we arrive at an expression which gives us the necessary total duration of stress to produce thrombosis. According to that equation, even in the absence of stress, any individual if he lives long enough should eventually develop a thrombosis. If, however the thrombokinase is destroyed, then it is necessary that during a sufficiently short time enough effective collisions occur to increase the amount of thrombokinase to a danger level. With this picture we find only theprobability of a formation of a thrombus as a function of the intensity of stress and its duration. The probability remains finite event for zero stress, showing that even individuals who are always relaxed may develop thrombi. In the second part of the paper an outline of a biochemical approach is given. Inasmuch as we know very little about the biochemical mechanism involved, it is simply assumed that during a stress of intensityS and duration Δ there is a probability of a thrombosis occurring. Then by applying the theory of probabilities, an expression is derived giving the probability of occurrence of a thrombus after a given number of stress incidences. The expressions are different from those obtained from the first approach but lead basically to the same results. A comparison of both or of their combination, with available data may give indications as to which of the factors plays a preponderant part.     

Evaluation of the kinetic parameters of the activation of trypsinogen by trypsin

Kinetic analysis of the mechanism of trypsinogen activation by trypsin under rapid equilibrium conditions and certain relationships between the rate constants are presented. The kinetic equations are valid from the beginning of the reaction. In addition, we suggest a procedure, based on the above equations, for the evaluation of the kinetic parameters of the reaction. This procedure is applied to a set of experimental data collected during the activation of bovine trypsinogen by trypsin at 30 degrees C (pH 8.1) in 0.01 M CaCl2. In this system, the amount of active enzyme increases exponentially, as expected from an autocatalytic process. The apparent rate constant, delta, governing this increase would vary linearly with the trypsinogen concentration, [Z]0, if no Michaelis complex was detectable. However, the increase in delta with [Z]0 is clearly non-linear and fits a hyperbola (delta = k2[Z]0/(Kz + [Z]0)) well.     

Meizothrombin formation during factor Xa-catalyzed prothrombin activation. Formation in a purified system and in plasma

Meizothrombin and thrombin formation were quantitated during factor Xa-catalyzed activation of human prothrombin in reaction systems containing purified proteins and in plasma. In the purified system considerable amounts of meizothrombin accumulated when prothrombin was activated by factor Xa (with or without accessory components) under initial steady state conditions. The ratio of the rates of meizothrombin and thrombin formation was not influenced by variation of the pH, temperature, or ionic strength of the reaction medium. When 2 microM prothrombin was activated by the complete prothrombinase complex (factor Xa, factor Va, Ca2+, and phospholipid) 80-90% of the initially formed reaction product was meizothrombin. Lowering the prothrombin concentration from 2 to 0.03 microM caused a gradual decrease in the ratio of meizothrombin/thrombin formation from 5 to 0.6. When the phosphatidylserine content of the phospholipid vesicles was varied between 20 and 1 mol % and prothrombin activation was analyzed at 2 microM prothrombin the relative amount of meizothrombin formed decreased from 85 to 55%. With platelets, cephalin, or thromboplastin as procoagulant lipid, thrombin was the major reaction product and only 30-40% of the activation product was meizothrombin. We also analyzed complete time courses of prothrombin activation both with purified proteins and in plasma. In reaction systems with purified proteins substantial amounts of meizothrombin accumulated under a wide variety of experimental conditions. However, little or no meizothrombin was detected in plasma in which coagulation was initiated via the extrinsic pathway with thromboplastin or via the intrinsic pathway with kaolin plus phospholipid (cephalin, platelets, or phosphatidylserine-containing vesicles). Thus, thrombin was the only active prothrombin activation product that accumulated during ex vivo coagulation experiments in plasma.     

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.     

Theoretical calculations on hydrogenase kinetics: explanation of the lag phase and the enzyme concentration dependence of the activity of hydrogenase uptake

Two models of the hydrogenase reaction cycle were investigated by means of theoretical calculations and model simulations. The first model is the widely accepted triangular hydrogenase reaction cycle with minor modifications; the second is a modified triangular model, where we have introduced an autocatalytic step into the reaction cycle. Both models include a one-step activation reaction. The theoretical calculations and model simulations corroborate the assumed autocatalytic reaction step concluded from the experimental characteristics of the hydrogenase reaction.     

Proposed kinetic mechanism of the production of biodiesel from palm oil using lipase

Experimental determination of the separate effects of palm oil and methanol concentrations on the rate of their enzymatic transesterification was used to propose suitable mechanismic steps and to test the generated kinetic model. The reaction took place in n-hexane organic medium and the lipase used was from Mucor miehei. At a constant methanol concentration of 300 mol m⁻³, it was found that, initially as the palm oil concentration increased, the initial reaction rate increased. However, the initial rate dropped sharply at substrate concentrations larger than 1250 mol m⁻³. Similar behaviour was observed for methanol concentration effect, where at a constant substrate concentration of 1000 mol m⁻³, the initial rate of reaction dropped at methanol concentrations larger than 3000 mol m⁻³. Ping Pong Bi Bi mechanism with inhibition by both reactants was adopted as it best explains the experimental findings. A mathematical model was developed from a proposed kinetic mechanism and was used to identify the regions where the effect of inhibition by both substrates arised. The proposed model equation is essential for predicting the rate of methanolysis of palm oil in a batch or a continuous reactor and for determining the optimal conditions for biodiesel production.     

An autocatalytic step in the reaction cycle of hydrogenase from Thiocapsa roseopersicina can explain the special characteristics of the enzyme reaction

A moving front has been observed as a special pattern during the hydrogenase-catalyzed reaction of hydrogen uptake with benzyl viologen as electron acceptor in a thin-layer reaction chamber. Such fronts start spontaneously and at random times at different points of the reaction chamber; blue spheres are seen expanding at constant speed and amplitude. The number of observable starting points depends on the hydrogenase concentration. Fronts can be initiated by injecting either a small amount of completed reaction mixture or activated hydrogenase, but not by injecting a low concentration of reduced benzyl viologen. These characteristics are consistent with an autocatalytic reaction step in the enzyme reaction. The special characteristics of the hydrogen-uptake reaction in the bulk reaction (a long lag phase, and the enzyme concentration dependence of the lag phase) support the autocatalytic nature. We conclude that there is at least one autocatalytic reaction step in the hydrogenase-catalyzed reaction. The two possible autocatalytic schemes for hydrogenase are prion-type autocatalysis, in which two enzyme forms interact, and product-activation autocatalysis, where a reduced electron acceptor and an inactive enzyme form interact. The experimental results strongly support the occurrence of prion-type autocatalysis.     

The anticoagulant property of a lipoid anticoagulant and the mechanism of its influence on hemocoagulation]

A lipoid anticoagulant (LA) from human brain tissue was shown to possess a higher anticoagulant activity than previously obtained preparations. It has been established that out of four phospholipids present in LA, only phosphatidyl serine inhibits the coagulant activity of plasma. In isolated hemocoagulating systems LA and phosphatidyl serine were shown to inhibit prothrombin conversion catalyzed by thrombokinase and to exert the antithrombic action in the system thrombin--fibrinogen. Kinetic study of phosphatidyl serine- and LA-induced inhibition of thrombine formation and the thrombine--fibrinogen reaction by conjugated inhibition. Similar kinetic behaviour, observed upon inhibition of both processes by phosphatidyl serine and LA suggest that phosphatidyl serine is a main anticoagulant agent of LA.     

The autocatalytic step is an integral part of the hydrogenase cycle

We earlier proved the involvement of an autocatalytic step in the oxidation of H₂ by HynSL hydrogenase from Thiocapsa roseopersicina, and demonstrated that two enzyme forms interact in this step. Using a modified thin-layer reaction chamber which permits quantitative analysis of the concentration of the reaction product (reduced benzyl viologen) in the reaction volume during the oxidation of H₂, we now show that the steady-state concentration of the product displays a strong enzyme concentration dependence. This experimental fact can be explained only if the previously detected autocatalytic step occurs inside the catalytic enzyme-cycle and not in the enzyme activation process. Consequently, both interacting enzyme forms should participate in the catalytic cycle of the enzyme. As far as we are aware, this is the first experimental observation of such a phenomenon resulting in an apparent inhibition of the enzyme. It is additionally concluded that the interaction of the two enzyme forms should result in a conformational change in the enzyme–substrate form. This scheme is very similar to that of prion reactions. Since merely a few molecules are involved at some point of the reaction, this process is entirely stochastic in nature. We have therefore developed a stochastic calculation method, calculations with which lent support to the conclusion drawn from the experiment.     

Modulation of human prothrombin activation on phospholipid vesicles and platelets using monoclonal antibodies to prothrombin fragment 2

Prothrombin contains two kringle domains that are removed during activation to the blood clotting enzyme alpha-thrombin. By analogy with other kringle-containing proteins the prothrombin kringles may play a role in the protein-protein interactions necessary for prothrombin activation. Four monoclonal antibodies to prothrombin kringle 2 have been produced against human prothrombin, and a fifth monoclonal antibody was produced against a synthetic peptide consisting of amino acid residues 216-231 of kringle 2. Each antibody was tested for its ability to block prothrombin activation by factor Xa. In the presence of phosphatidylcholine/phosphatidylserine vesicles and factor Va, two of the antibodies, alpha HII-3 and alpha HII-4, inhibited prothrombin activation at a 90 and 50% level, respectively. Two other monoclonal antibodies (alpha HII-6 and alpha HII-7) and the antipeptide antibody (alpha HII-5) had no effect on prothrombin activation. When factor Xa was the catalyst alone, antibody alpha HII-3 lost the ability to inhibit prothrombin activation whereas antibody alpha HII-4 again partially inhibited the reaction. When human platelets were the reaction surface, the patterns of inhibition by the anti-fragment 2 antibodies were identical to that observed with phospholipid vesicles. These data suggest a role for prothrombin fragment 2 in activation, possibly by mediating the interaction of substrate prothrombin with factor Xa or factor Va on the phospholipid surface.     

Chromatography of blood-clotting factors and serum proteins on columns of diatomaceous earth

1. In batch adsorptions with prothrombin solutions, hyflo was the weakest adsorbent, standard super-cel intermediate, and filter-cel strongest. Of these three grades of diatomaceous earth, hyflo has the smallest surface area per gram and filter-cel the largest. In parallel breakthrough experiments, a column of standard super-cel had a capacity almost six times that of a hyflo column. 2. After partial removal of impurities by diatomaceous earth, prothrombin preparations contained less thrombokinase, were more stable, and displayed less tendency to form thrombin "spontaneously." Thrombokinase (or its precursor) was removed from a preparation of prothrombin by passage through a filter cake of standard super-cel. The specific activity of the prothrombin was increased; and 62 per cent of the activity was recovered. 3. Prothrombin was adsorbed from an ammonium sulfate solution at pH 5.26 by columns of hyflo or standard super-cel. When eluted by phosphate solutions, the protein moved down the columns more readily at higher pH and higher concentration of phosphate salts, within the pH range 5.0 to 6.6, and within the phosphate range 0.1 to 1.0 M. 4. Thrombin was adsorbed on a column of standard super-cel at pH 5.11. As successive eluents passed through the column, the thrombin emerged between two bands of impurities. The specific activity of the thrombin was raised; and 83 per cent of the activity was recovered. 5. With a column of standard super-cel, and with a series of eluents within the pH range 5.1 to 6.3, total serum proteins were separated into four major bands. About 94 per cent of the protein was recovered.     

Kinetics of autocatalytic zymogen activation measured by a coupled reaction: pepsinogen autoactivation

A kinetic study was performed of a model for an autocatalytic zymogen activation process involving both intra- and intermolecular routes, to which a chromogenic reaction in which the active enzyme acts upon one of its substrates was coupled to continuously monitor the reaction. Kinetic equations describing the evolution of species involved in the system with time were obtained. These equations are valid for any zymogen autocatalytic activation process under the same initial conditions. Experimental design and kinetic data analysis procedures to evaluate the kinetic parameters, based on the derived kinetic equations, are suggested. In addition, a dimensionless distribution coefficient was defined, which shows mathematically whether the intra- or the intermolecular route prevails once the kinetic parameters involved in the system are known. The validity of the results obtained was checked using simulated curves for the species involved. As an example of application of the method, the system is experimentally illustrated by the continuous monitoring of pepsinogen transformation to pepsin.     

Conditions for chiral asymmetry generation in the preparation of the chiral octahedral cobalt complex

We have reported that the random chiral asymmetry generation, which is a spontaneous preferential generation of one enantiomer, was observed in the synthesis of a chiral octahedral cobalt complex, cis-[CoBr(NH(3))(en)(2)]Br(2). In this article, we review our studies to explain in this system the autocatalytic growth of small enantiomeric excess that arises due to statistical fluctuations. One important experimental finding was that the rate of chiral autocatalysis increased with increasing the degree of supersaturation. Furthermore, our numerical simulation indicates that even small inhomogeneities in the reaction system may play a significant role because their effect is amplified by the autocatalytic reaction under appropriate conditions. In a small volume, fluctuations in concentration can grow if the autocatalytic growth overcomes the diffusional loss of the excess concentration from this volume. This may makes the enantiomeric excess of the chiral complex randomly fluctuate from run to run.     

Autoacylation of myelin proteolipid protein with acyl coenzyme A

Rat brain myelin proteolipid protein (PLP) is known to contain long chain, covalently bound fatty acids. In the course of characterizing the mechanism of acylation, we found that the isolated PLP, in the absence of any membrane fraction, was esterified after incubation with [3H]palmitoyl coenzyme A (CoA). This observation demonstrated that the protein acts as both an acylating enzyme and an acceptor. Thus, acylation occurs by an autocatalytic process. The possibility of a separate acyltransferase that copurifies with PLP was essentially excluded by adding brain subcellular fractions to the reaction mixtures and by changing the isolation procedure. After deacylation, the protein was acylated at a 4-fold greater rate, suggesting that the original sites were reacylated. The palmitoyl-CoA concentration followed Michaelis kinetics, confirming that spontaneous acylation was not occurring. Pulse-chase experiments indicated that the reaction entails net addition of acyl groups. Although fatty acids are bound via an O-ester linkage, free SH groups are required in the reaction. Denaturation of the protein by sodium dodecyl sulfate or heat inhibits the reaction, whereas cerulenin has little or no effect. PO, the major protein in peripheral nerve myelin, is also an acylated protein, but it was not labeled upon incubation of either peripheral myelin or the isolated protein with [3H]palmitoyl-CoA, demonstrating that it is acylated by a different route. Several synthetic peptides derived from PLP sequences with sites known to be acylated in vivo as well as a series of deacylated PLP tryptic peptides were not labeled, indicating that integrity of the protein is required for acylation. Limited proteolysis and peptide mapping showed that the same sites are acylated in vitro or in vivo, suggesting that the autocatalytic acylation reaction is physiological.     

Characterization of the interaction between factor Xa and bovine aortic endothelial cells

Cultured bovine aortic endothelial cells incubated with Factor Xa activate prothrombin. Factor V, synthesized by the endothelial cells, or plasma Factor V and calcium are required for the reaction. In the present study, it has been demonstrated that 125I-Factor Xa binds specifically to endothelial cells. In addition, the activation of prothrombin by Factor Xa and aortic endothelial cells has been further characterized. The binding of 125I-Factor Xa to endothelial cells was saturable and reversible. The equilibrium dissociation constant (Kd) for 125I-Factor Xa binding was 3.6 X 10(-9) M, with 39000 molecules bound per cell. 125I-Factor Xa, inactivated by diisopropylfluorophosphate did not bind specifically to endothelial cells, indicating that the active site of Factor Xa was required for binding. Factor Xa, but not activated protein C, competed with 125I-Factor Xa for binding. Autoradiograms of sodium dodecyl sulfate-polyacrylamide gels of cell lysates indicated that the radiolabeled material that bound to the cells had electrophoretic mobility identical to Factors Xa alpha and Xa beta. Although Factor X partially inhibited the binding of 125I-Factor Xa, Factor Xa did not inhibit the binding of 125I-Factor X, indicating that the zymogen and enzyme bound to different receptors. The relationship of the 125I-Factor Xa binding which was measured in these studies to aortic endothelial cell prothrombin activation is unclear since an anti-Factor V IgG blocked prothrombin activation but not Factor Xa binding. Additionally, 125I-Factor Xa binds to nonvascular cells; these cells do not activate prothrombin in the presence of Factor Xa. Moreover, the calcium requirements for each reaction and the saturation curves of 125I-Factor Xa binding and prothrombin activation differ. Although these data do not exclude a relationship between Factor Xa binding and prothrombin activation, the binding of 125I-Factor Xa to aortic endothelium measured in these studies may be related to a separate cellular function. To further characterize prothrombin activation by Factor Xa and endothelial cells, the rates of thrombin generation by intact bovine aorta or endothelial cells derived from this tissue were compared and were found to be equivalent. These data indicate that vascular endothelium may serve as a physiologic surface for hemostasis.     

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.