RESUMEN
Herein, a novel electrochemical glucose biosensor based on glucose oxidase (GOx) immobilized on a surface containing platinum nanoparticles (PtNPs) electrodeposited on poly(Azure A) (PAA) previously electropolymerized on activated screen-printed carbon electrodes (GOx-PtNPs-PAA-aSPCEs) is reported. The resulting electrochemical biosensor was validated towards glucose oxidation in real samples and further electrochemical measurement associated with the generated H2O2. The electrochemical biosensor showed an excellent sensitivity (42.7 µA mM-1 cm-2), limit of detection (7.6 µM), linear range (20 µM-2.3 mM), and good selectivity towards glucose determination. Furthermore, and most importantly, the detection of glucose was performed at a low potential (0.2 V vs. Ag). The high performance of the electrochemical biosensor was explained through surface exploration using field emission SEM, XPS, and impedance measurements. The electrochemical biosensor was successfully applied to glucose quantification in several real samples (commercial juices and a plant cell culture medium), exhibiting a high accuracy when compared with a classical spectrophotometric method. This electrochemical biosensor can be easily prepared and opens up a good alternative in the development of new sensitive glucose sensors.
Asunto(s)
Colorantes Azulados , Técnicas Biosensibles , Glucosa/análisis , Nanopartículas del Metal , Carbón Orgánico , Técnicas Electroquímicas , Electrodos , Enzimas Inmovilizadas , Glucosa Oxidasa , Peróxido de Hidrógeno , Platino (Metal)RESUMEN
The use of disposable screen-printed electrodes (SPEs) has extraordinarily grown in the last years. In this paper, conductive inks from scrapped SPEs were removed by acid leaching, providing high value feedstocks suitable for the electrochemical deposition of Ag, Pt and Ag core-Pt shell-like bimetallic (AgPt) nanoparticles, onto screen-printed carbon electrodes (ML@SPCEs, M = Ag, Pt or AgPt, L = metal nanoparticles from leaching solutions). ML@SPCEs were characterized by scanning electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy. The results were compared to those obtained when metal nanoparticles were synthesised using standard solutions of metal salts (MS@SPCEs). Both ML@SPCEs and MS@SPCEs exhibited similar cyclic voltammetric patterns referred to the electrochemical stripping of silver or the adsorption/desorption of hydrogen/anions in the case of platinum, proving leaching solutions extremely effective for the electrodeposition of metallic nanoparticles. The use of both ML@SPCEs and MS@SPCEs proved effective in enhancing the sensitivity for the detection of H2O2 in phosphate buffer solutions (pH = 7). The AgPtL@SPCE was used as proof of concept for the validation of an amperometric sensor for the determination of H2O2 within laundry boosters and antiseptic samples. The electrochemical sensor gave good agreement with the results obtained by a spectrophotometric method with H2O2 recoveries between 100.6% and 106.4%.
RESUMEN
Hydrogen peroxide triggers a redox cycle between methemoglobin and ferrylhemoglobin, leading to protein inactivation and oxygen evolution. In the present paper, the catalase-like oxygen production by human methemoglobin in the presence of H(2)O(2) was kinetically characterized with a Clark-type electrode. Progress curves showed a pseudo-steady state in the first minutes of the reaction, while double-reciprocal plots were upwardly concave, indicating positive co-operativity dependent upon protein concentration, which is a very unusual kinetic behavior. Addition of superoxide radical scavengers slightly increased activity, suggesting that most oxygen was produced biocatalytically. By considering all the experimental data obtained, a possible mechanism was proposed, including: (a) competition between the one-electron and the two-electron reductions of the oxoferryl free radical species of hemoglobin, giving rise to ferrylhemoglobin and methemoglobin, respectively; (b) competition between the superoxide-dependent inactivation of the protein and its reduction back to the met state. Computer simulations of that model have been performed by numerically integrating the differential equations set describing the mechanism, which was seen to yield predictions of the kinetic parameters variation consistently with the kinetic behavior experimentally observed. We suggest that the catalase-like activity of methemoglobin must predominantly be a biocatalytic reaction that protects the protein against H(2)O(2)-induced suicide inactivation.
Asunto(s)
Catalasa/metabolismo , Peróxido de Hidrógeno/metabolismo , Metahemoglobina/metabolismo , Electrodos , Depuradores de Radicales Libres/metabolismo , Humanos , Cinética , Metamioglobina/metabolismo , Oxígeno/metabolismo , Oxihemoglobinas/metabolismo , Espectrofotometría , Superóxidos/metabolismoRESUMEN
Hydroperoxides play important roles in atmospheric chemical processes since they act as strong oxidants. This paper details with the modification, characterization and performance of different carbon-based screen-printed electrodes to develop a sensor that allows to analyze organic and inorganic hydroperoxides in atmospheric samples. Commercial electrodes made up of graphite, graphene, carbon nanotubes and graphene oxide were electrochemically activated and subsequently modified by layer-by-layer method with a conducting polymer of azure-A and electrodeposited platinum nanoparticles. Characterization of modified electrodes was performed by FE-SEM, XPS, Raman spectroscopy, cyclic voltammetry, and impedance spectroscopy. Even though all modified carbonaceous substrates enabled the development of competitive electrochemical sensors for (in)organic hydroperoxides detection, carbon nanotubes underlying substrate exhibited the best performances in terms of sensitivity, stability, limit of detection and linear range. This amperometric sensor displayed linear responses to hydroperoxides over 0.081-450 µM with detection limits in the range of 24-558 nM and sensitivity values among 0.0628±1.6E-4 and 0.0112±0.71E-4 µA/µM for the different hydroperoxides herein studied. The developed electrochemical sensor was successfully applied to the analysis of (in)organic hydroperoxides in rainwater samples. Measurements in rainwater were performed in a city located in the East of Spain and collected at two different sites (downtown and suburban area) on two different dates (July and November 2020). The presented results demonstrated high sensitivity and selectivity for the detection of hydroperoxides among a plethora of substances naturally present in rainwater.
Asunto(s)
Grafito , Nanopartículas del Metal , Nanotubos de Carbono , Técnicas Electroquímicas , Electrodos , Peróxido de Hidrógeno , Límite de Detección , Platino (Metal)RESUMEN
Monitoring of hydrogen peroxide (H2O2) in living cells has high significance for understanding its functions. We herein report an enzymeless H2O2 sensor consisting of a previously activated screen-printed carbon electrode modified with Pt nanoparticles electrogenerated on a supporting conductive layer of polyazure A-dodecyl sulfate. This electrode was used to investigate the dynamic process of H2O2 release from living grapevine cells under different (a)biotic stresses. The modified surfaces were characterized by FESEM/EDX, EIS and cyclic voltammetry. Sensor analytical performance was studied in a cell culture medium under aerobic conditions, as required for cell survival. In relation to the synergistic effect between the metal nanoparticles and the conjugated polymer, this electrode showed good stability, excellent analytical performance combined with a rapid response (<2s) and limit of detection of 24.9 nM in the culture medium. The modified electrodes could fulfill the real-time measurement requirement of H2O2 release from living plant cells to the extracellular medium operating continuously, even in experiments lasting more than 12 h. Methyl jasmonate, L-methionine, clopyralid and the fungus Botrytis cinerea were the eliciting agents chosen to induce oxidative stress in the plant cells. This work demonstrates the huge potential of this sensor for the real-time tracking of the H2O2 released from living cells under different physiological conditions.
Asunto(s)
Colorantes Azulados/química , Técnicas Biosensibles/instrumentación , Peróxido de Hidrógeno/metabolismo , Nanopartículas del Metal/química , Células Vegetales/metabolismo , Platino (Metal)/química , Impresión , Botrytis/fisiología , Carbono/química , Electroquímica , Electrodos , Límite de Detección , Células Vegetales/microbiologíaRESUMEN
Oxidation of acetaminophen by human methemoglobin in the presence of H(2)O(2) has been kinetically studied in the present paper. The drug showed a protective effect against the H(2)O(2)-induced irreversible inactivation of the protein, thus indicating the competition among both ligands, H(2)O(2) and acetaminophen for the protein. The stoichiometry of the reaction is variable and depends on relative initial concentrations of H(2)O(2) and the drug owing to their competitive behavior. In addition and unexpectedly, the protein exhibits non Michaelian kinetics against both acetaminophen and H(2)O(2) under steady-state conditions and shows negative co-operativity with Hill coefficients in the 0.3-0.7 range. Therefore, these data were compared to those obtained with myoglobin under similar experimental conditions, and the same results were observed. This led us to propose a mechanism for the peroxidase-like activity of hemoglobin, which accounts for the experimental results obtained herein. The steady-state rate equation for this mechanism has been obtained and is also consistent with the experimental data, thus indicating the goodness of the model proposed herein. The results presented in this work provide new insights into the oxidation mechanism of acetaminophen.
Asunto(s)
Acetaminofén/química , Metahemoglobina/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Cinética , Oxidación-Reducción , Peroxidasa/metabolismo , Espectrofotometría UltravioletaRESUMEN
In the present paper, poly(azure A) (PAA) films were electrosynthetized in the presence of different doping anions on disposable screen-printed carbon electrodes (SPCEs). The anions used included inorganic monoatomic (chloride and fluoride), inorganic polyatomic (nitrate and sulfate) and organic polyatomic (dodecyl sulfate, DS) species. The coated electrodes thus obtained were characterized by electrochemical techniques and SEM. They showed improved electrocatalytic activities towards hydrogen peroxide oxidation compared to that of a bare SPCE. In particular, the insertion of DS anions inside PAA films provided a special sensitivity to the electrocatalysis of H2O2, which endowed these electrodes with promising analytical features for H2O2 quantification. We obtained a wide linear response for H2O2 within a range of 5 µM to 3 mM and a limit of detection of 1.43 ± 0.10 µM (signal-to-noise ratio of 3). Furthermore, sensitivity was 72.4 ± 0.49 nA·µM-1âcm-2 at a relatively low electrocatalytic oxidation overpotential of 0.5 V vs. Ag. The applicability of this boosted system was tested by the analysis of H2O2 in commercial samples of a hair lightener and an antiseptic and was corroborated by spectrophotometric methods.
RESUMEN
In the present paper, a kinetic study is made of the behavior of a moiety-conserved ternary cycle between the adenine nucleotides. The system contains the enzymes S-acetyl coenzyme A synthetase, adenylate kinase and pyruvate kinase, and converts ATP into AMP, then into ADP and finally back to ATP. L-Lactate dehydrogenase is added to the system to enable continuous monitoring of the progress of the reaction. The cycle cannot work when the only recycling substrate in the reaction medium is AMP. A mathematical model is proposed whose kinetic behavior has been analyzed both numerically by integration of the nonlinear differential equations describing the kinetics of the reactions involved, and analytically under steady-state conditions, with good agreement with the experimental results being obtained. The data obtained showed that there is a threshold value of the S-acetyl coenzyme A synthetase/adenylate kinase ratio, above which the cycle stops because all the recycling substrate has been accumulated as AMP, never reaching the steady state. In addition, the concept of adenylate energy charge has been applied to the system, obtaining the enabled values of the rate constants for a fixed adenylate energy charge value and vice versa.
Asunto(s)
Nucleótidos de Adenina/metabolismo , Cromatografía Líquida de Alta Presión , Simulación por Computador , Cinética , Modelos TeóricosRESUMEN
Taking as the starting point a recently suggested reaction scheme for zymogen activation involving intra- and intermolecular routes and the enzyme-zymogen complex, we carry out a complete analysis of the relative contribution of both routes in the process. This analysis suggests the definition of new dimensionless parameters allowing the elaboration, from the values of the rate constants and initial conditions, of the time course of the contribution of the two routes. The procedure mentioned above related to a concrete reaction scheme is extrapolated to any other model of autocatalytic zymogen activation involving intra- and intermolecular routes. Finally, we discuss the contribution of both of the activating routes in pepsinogen activation into pepsin using the values of the kinetic parameters given in the literature.
Asunto(s)
Algoritmos , Precursores Enzimáticos/metabolismo , Pepsinógeno A/metabolismo , Catálisis , Activación Enzimática , Precursores Enzimáticos/química , Cinética , Modelos Químicos , Pepsinógeno A/químicaRESUMEN
BACKGROUND: Light/dark cycles are probably the most important environmental signals that regulate plant development. Light is essential for photosynthesis, but an excess, in combination with the unavoidable presence of atmospheric oxygen inside the chloroplast, leads to excessive reactive oxygen species production. Among the defense mechanisms that activate plants to cope with environmental stress situations, it is worth noting the ascorbate-glutathione cycle, a complex metabolic pathway in which a variety of photochemical, chemical and enzymatic steps are involved. RESULTS: We herein studied the dynamic behavior of this pathway under light/dark conditions and for several consecutive days. For this purpose, a mathematical model was developed including a variable electron source with a rate law proportional to the intensity of solar irradiance during the photoperiod, and which is continuously turned off at night and on again the next day. The model is defined by a nonlinear system of ordinary differential equations with an on/off time-dependent input, including a parameter to simulate the fact that the photoperiod length is not constant throughout the year, and which takes into account the particular experimental kinetics of each enzyme involved in the pathway. Unlike previous models, which have only provided steady-state solutions, the present model is able to simulate diurnal fluctuations in the metabolite concentrations, fluxes and enzymatic rates involved in the network. CONCLUSIONS: The obtained results are broadly consistent with experimental observations and highlight the key role played by ascorbate recycling for plants to adapt to their surrounding environment. This approach provides a new strategy to in vivo studies to analyze plant defense mechanisms against oxidative stress induced by external changes, which can also be extrapolated to other complex metabolic pathways to constitute a useful tool to the scientific community in general.
Asunto(s)
Ácido Ascórbico/metabolismo , Cloroplastos/metabolismo , Cloroplastos/efectos de la radiación , Oscuridad , Glutatión/metabolismo , Modelos Biológicos , Antioxidantes/metabolismo , Relación Dosis-Respuesta en la Radiación , NADP/metabolismo , Estrés Oxidativo/efectos de la radiación , Fotosíntesis/efectos de la radiación , Plantas/metabolismo , Plantas/efectos de la radiaciónRESUMEN
A mathematical description was made of an autocatalytic zymogen activation mechanism involving both intra- and intermolecular routes. The reversible formation of an active intermediary enzyme-zymogen complex was included in the intermolecular activation route, thus allowing a Michaelis-Menten constant to be defined for the activation of the zymogen towards the active enzyme. Time-concentration equations describing the evolution of the species involved in the system were obtained. In addition, we have derived the corresponding kinetic equations for particular cases of the general model studied. Experimental design and kinetic data analysis procedures to evaluate the kinetic parameters, based on the derived kinetic equations, are suggested. The validity of the results obtained were checked by using simulated progress curves of the species involved. The model is generally good enough to be applied to the experimental kinetic study of the activation of different zymogens of physiological interest. The system is illustrated by following the transformation kinetics of pepsinogen into pepsin.
Asunto(s)
Precursores Enzimáticos/metabolismo , Pepsinógeno A/metabolismo , Animales , Cinética , Reproducibilidad de los Resultados , PorcinosRESUMEN
This paper presents a kinetic analysis of the whole reaction course, i.e. of both the transient phase and the steady state, of open multicyclic enzyme cascade systems. Equations for fractional modifications are obtained which are valid for the whole reaction course. The steady state expressions for the fractional modifications were derived from the latter equations since they are not restricted to the condition of rapid equilibrium. Finally, the validity of our results is discussed and tested by numerical integration. Apart from the intrinsic value of knowing the kinetic behaviour of any of the species involved in any open multicyclic enzyme cascade, the kinetic analysis presented here can be the basis of future contributions concerning open multicyclic enzyme cascades which require the knowledge of their time course equations (e.g. evaluation of the time needed to reach the steady state, suggestion of kinetic data analysis, etc.), analogous to those already carried out for open bicyclic cascades.
Asunto(s)
Enzimas/metabolismo , Simulación por Computador , Cinética , Modelos Teóricos , Reproducibilidad de los ResultadosRESUMEN
3'-Hydroxyacetaminophen, a catechol metabolite of N-acetyl-p-aminophenol (acetaminophen) and N-acetyl-m-aminophenol (a structural analogue of acetaminophen and considered as a possible alternative because it is not hepatotoxic), is enzymatically synthesized for the first time using mushroom tyrosinase. Although reported to be weakly hepatotoxic in vivo, this catechol derivative of acetaminophen is not commercially available. This compound was obtained from its monophenolic precursor, acetaminophen, using the enzyme tyrosinase in the presence of an excess of ascorbic acid, thus reducing back the o-quinone product of catalytic activity to the catechol acetaminophen derivative. A mathematical model of the system is proposed, which is also applicable to the tyrosinase-mediated synthesis of any o-diphenolic compound from its corresponding monophenol. This synthesis procedure is continuous, easy to perform and control, and adaptable to a bioreactor with the immobilized enzyme for industrial purposes in a nonpolluting way.
Asunto(s)
Acetaminofén/química , Acetaminofén/síntesis química , Agaricales/enzimología , Ácido Ascórbico/química , Modelos Químicos , Monofenol Monooxigenasa/química , Acetaminofén/análogos & derivados , Catálisis , Simulación por Computador , Enzimas Inmovilizadas/química , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Biochemical energy is the fundamental element that maintains both the adequate turnover of the biomolecular structures and the functional metabolic viability of unicellular organisms. The levels of ATP, ADP and AMP reflect roughly the energetic status of the cell, and a precise ratio relating them was proposed by Atkinson as the adenylate energy charge (AEC). Under growth-phase conditions, cells maintain the AEC within narrow physiological values, despite extremely large fluctuations in the adenine nucleotides concentration. Intensive experimental studies have shown that these AEC values are preserved in a wide variety of organisms, both eukaryotes and prokaryotes. Here, to understand some of the functional elements involved in the cellular energy status, we present a computational model conformed by some key essential parts of the adenylate energy system. Specifically, we have considered (I) the main synthesis process of ATP from ADP, (II) the main catalyzed phosphotransfer reaction for interconversion of ATP, ADP and AMP, (III) the enzymatic hydrolysis of ATP yielding ADP, and (IV) the enzymatic hydrolysis of ATP providing AMP. This leads to a dynamic metabolic model (with the form of a delayed differential system) in which the enzymatic rate equations and all the physiological kinetic parameters have been explicitly considered and experimentally tested in vitro. Our central hypothesis is that cells are characterized by changing energy dynamics (homeorhesis). The results show that the AEC presents stable transitions between steady states and periodic oscillations and, in agreement with experimental data these oscillations range within the narrow AEC window. Furthermore, the model shows sustained oscillations in the Gibbs free energy and in the total nucleotide pool. The present study provides a step forward towards the understanding of the fundamental principles and quantitative laws governing the adenylate energy system, which is a fundamental element for unveiling the dynamics of cellular life.
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Adenosina Trifosfato/metabolismo , Metabolismo Energético , Homeostasis , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Animales , Simulación por Computador , Humanos , Cinética , Modelos Biológicos , PeriodicidadRESUMEN
The present paper describes the synthesis and characterization of a new polymeric biomaterial mineralized with calcium phosphate using the reaction-diffusion method. The scaffold of this biomaterial was a hydrogel constituted by biocompatible polyethylene glycol methyl ether methacrylate (PEGMEM) and 2-(dimethylamino)ethyl methacrylate (DMAEM), which were cross-linked with N-N'-methylenebisacrylamide (BIS). The cross-linking content of the hydrogels was varied from 0.25% to 15% (w/w). The gels were used as matrix where two reactants (Na2HPO4 and CaCl2) diffused from both ends of the gel and upon encountering produced calcium phosphate crystals that precipitated within the polymer matrix forming bands. The shape of the crystals was tuned by modifying the matrix porosity in such a way that when the polymer matrix was slightly reticulated the diffusion reaction produced round calcium phosphate microcrystals, whilst when the polymer matrix was highly reticulated the reaction yielded flat calcium phosphate crystals. Selected area electron diffraction performed on the nanocrystals that constitute the microcrystals showed that they were formed by Brushite (CaHPO4.2H2O). This new composite material could be useful in medical and dentistry applications such as bone regeneration, bone repair or tissue engineering.
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Materiales Biocompatibles/química , Fosfatos de Calcio/química , Hidrogeles/química , Metacrilatos/química , Polietilenglicoles/química , Fosfatos de Calcio/metabolismo , Ensayo de MaterialesRESUMEN
In the present work, 13 p-substituted phenols with different functional groups have been systematically evaluated as metHb substrates by means of HPLC analysis. Non-hyperbolic kinetics were observed and Hill coefficients in the 0.37-1.00 range were obtained. The catalytic constants and the Hill coefficients were found to be quantitatively correlated with two independent variables: the energy level of the highest-occupied molecular orbital (E(HOMO)), which describes the intrinsic redox activity of the substrates and the pK(a)-values, which are related to substrate ionization. Oxygen evolution in the presence of each phenol derivative was also measured, and good correlation between peroxidase-like and catalase-like activities of the protein was observed. It is also shown that bovine metHb, although less active than other peroxidases, may represent a good alternative from an economical point of view for phenol removal processes. The equations here obtained may serve as a basis to further explore the potential use of metHb-mediated reactions in the treatment of phenols in wastewaters and to predict which phenol will be removed most efficiently under this treatment with satisfactory reliability.
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Peróxido de Hidrógeno/química , Metahemoglobina/química , Fenoles/aislamiento & purificación , Aguas Residuales/análisis , Contaminantes Químicos del Agua/aislamiento & purificación , Purificación del Agua/métodos , Animales , Biocatálisis , Bovinos , Cinética , Oxidación-Reducción , Fenoles/química , Relación Estructura-Actividad Cuantitativa , Contaminantes Químicos del Agua/químicaRESUMEN
The glutathione-ascorbate redox pathway in chloroplasts is a complex network of spontaneous, photochemical, and enzymatic reactions for detoxifying hydrogen peroxide. This article presents a comprehensive sensitivity analysis of the system. A model has been constructed to simulate oxidative stress conditions, enabling steady-state concentrations of the metabolites involved in the pathway and photochemical and enzymatic fluxes to be calculated. The model includes an electron source whose flux is distributed among three competitive routes (photogeneration of O2-, photoreduction of NADP+ to NADPH, and photoreduction of monodehydroascorbate to ascorbate) and that allows the simulation of variations in NADPH concentration with time. Each enzyme considered is introduced in the model, taking into account its particular catalytic mechanism, including the inactivation of ascorbate peroxidase in the presence of low-ascorbate concentrations. Computer simulations pointed to the great sensitivity of the system to the ratio among fluxes corresponding to ascorbate and NADPH photoproduction and NADPH consumption by the Calvin cycle. Under oxidative stress conditions, the model shows a sequential depletion of antioxidant power in chloroplasts in the order NADPH, glutathione, ascorbate and their recovery in the reverse order. Decreasing levels of glutathione reductase, ascorbate peroxidase, and superoxide dismutase led to the irreversible photoinactivation of ascorbate peroxidase and the subsequent increase in hydrogen peroxide concentration, preceded by a maximum in dehydroascorbate reductase activity.
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Ácido Ascórbico/metabolismo , Cloroplastos/metabolismo , Simulación por Computador , Glutatión/metabolismo , Antioxidantes/metabolismo , Cloroplastos/enzimología , Electrones , Cinética , Modelos Biológicos , Oxidación-Reducción , Estrés FisiológicoRESUMEN
In the present paper, a kinetic analysis of a general model for proenzyme activation, where the activating enzyme and also the activated one are reversibly inhibited in two steps by two different inhibitors, has been performed. The cases in which both inhibitors are the same, or in which the inhibition is irreversible (only one or the two inhibition routes) are treated as particular cases of the general model. In addition, the kinetic behaviour of many other proenzyme activation systems involving inhibition, particular cases of the reaction scheme under study, can be obtained. The total number of particular cases for the general model under study is 370, so this approach offers to the scientific community working in limited proteolysis regulation for the first time a method based on general solutions which only needs to be specified to their concrete problem of zymogen activation. Finally, new adimensional parameters are introduced, allowing the knowledgement, in the case that any of the inhibition routes is irreversible, the relative weight of both activation and irreversible inhibition routes.
Asunto(s)
Inhibidores Enzimáticos/metabolismo , Precursores Enzimáticos/metabolismo , Animales , Activación Enzimática , Precursores Enzimáticos/antagonistas & inhibidores , Matemática , Modelos Químicos , Péptido Hidrolasas/metabolismo , Factores de TiempoRESUMEN
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.
Asunto(s)
Precursores Enzimáticos/metabolismo , Pepsinógeno A/metabolismo , Activación Enzimática , CinéticaRESUMEN
A mathematical description has been made of an enzyme amplification mechanism involving the coupling of two substrate cycles. In this amplification system one of the noncycling products of a first substrate cycle acts as a trigger molecule that continuously feeds a second substrate cycle. Time-concentration equations describing the evolution of the species involved in the system have been obtained. The model is illustrated by the quantification of nanomolar levels of ADP (and/or ATP) in a continuous assay involving the enzymes L-lactate dehydrogenase and L-lactate oxidase to cycle the pyruvate accumulated in a first enzymatic cycle constituted by the enzymes pyruvate kinase and hexokinase. Progress curves were seen to be parabolic, and, according to the kinetic equations obtained, followed second-order polynomials of the reaction time. Mathematical equations for minimizing the cost of the assays are also given. The model is applicable to the amplified analytical determination of low levels of a metabolite or an enzyme activity, and its amplification capacity, together with the simplicity of determining kinetic parameters, enable it to be employed in enzyme immunoassays to increase the magnitude of the measured response.