RESUMEN
Tyrosinase is a multi-copper enzyme which is widely distributed in different organisms and plays an important role in the melanogenesis and enzymatic browning. Therefore, its inhibitors can be attractive in cosmetics and medicinal industries as depigmentation agents and also in food and agriculture industries as antibrowning compounds. For this purpose, many natural, semi-synthetic and synthetic inhibitors have been developed by different screening methods to date. This review has focused on the tyrosinase inhibitors discovered from all sources and biochemically characterised in the last four decades.
Asunto(s)
Chalcona/farmacología , Cumarinas/farmacología , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Monofenol Monooxigenasa/antagonistas & inhibidores , Resveratrol/farmacología , Agaricales/enzimología , Animales , Chalcona/química , Cumarinas/química , Inhibidores Enzimáticos/química , Flavonoides/química , Humanos , Monofenol Monooxigenasa/metabolismo , Resveratrol/químicaAsunto(s)
Síndrome HELLP , Hemoperitoneo/etiología , Hepatopatías/etiología , Adulto , Femenino , Humanos , Embarazo , Rotura EspontáneaAsunto(s)
Humanos , Femenino , Embarazo , Adulto , Hemoperitoneo/complicaciones , Hemoperitoneo/diagnóstico , Síndrome HELLP/diagnóstico , Complicaciones del Embarazo/diagnóstico , Dexametasona/uso terapéutico , Hemoperitoneo/fisiopatología , Hemoperitoneo/terapia , Síndrome HELLP , Hematoma/complicacionesRESUMEN
Under aerobic or anaerobic conditions, tyrosinase undergoes a process of irreversible inactivation induced by its physiological substrate L-dopa. Under aerobic conditions, this inactivation occurs through a process of suicide inactivation involving the form oxy-tyrosinase. Under anaerobic conditions, both the met- and deoxy-tyrosinase forms undergo irreversible inactivation. Suicide inactivation in aerobic conditions is slower than the irreversible inactivation under anaerobic conditions. The enzyme has less affinity for the isomer D-dopa than for L-dopa but the velocity of inactivation is the same. We propose mechanisms to explain these processes.
Asunto(s)
Dihidroxifenilalanina/química , Monofenol Monooxigenasa/química , Agaricales/enzimología , Catálisis , Dominio Catalítico , Catecol Oxidasa/química , Cinética , Espectroscopía de Resonancia Magnética , Modelos Biológicos , Modelos Químicos , Oxígeno/química , Unión Proteica , Espectrofotometría/métodos , Factores de TiempoRESUMEN
There is controversy in the literature concerning the action of tetrahydropterines on the enzyme tyrosinase and on melanogenesis in general. In this study, we demonstrate that tetrahydropterines can inhibit melanogenesis in several ways: i) by non-enzymatic inhibition involving purely chemical reactions reducing o-dopaquinone to L-dopa, ii) by acting as substrates which compete with L-tyr and L-dopa, since they are substrates of tyrosinase; and iii) by irreversibly inhibiting the enzymatic forms met-tyrosinase and deoxy-tyrosinase in anaerobic conditions. Three tetrahydropterines have been kinetically characterised as tyrosinase substrates: 6-R-L-erythro-5,6,7,8-tetrahydrobiopterin, 6-methyl-5,6,7,8-tetrahydropterine and 6,7-(R,S)-dimethyl-5,6,7,8-tetrahydropterine. A kinetic reaction mechanism is proposed to explain the oxidation of these compounds by tyrosinase.
Asunto(s)
Melaninas/biosíntesis , Monofenol Monooxigenasa/antagonistas & inhibidores , Pterinas/farmacología , Agaricales/enzimología , Unión Competitiva , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Cinética , Levodopa/metabolismo , Monofenol Monooxigenasa/metabolismo , Oxidación-Reducción , Pterinas/química , Especificidad por Sustrato , Tirosina/metabolismoRESUMEN
INTRODUCTION: Laparoscopic resection of rectal cancer has been proven efficacious but morbidity and oncological outcome need to be investigated in a randomized clinical trial. TRIAL DESIGN: Non-inferiority randomized clinical trial. METHODS: The COLOR II trial is an ongoing international randomized clinical trial. Currently 27 hospitals from Europe, South Korea and Canada are including patients. The primary endpoint is loco-regional recurrence rate three years post-operatively. Secondary endpoints cover quality of life, overall and disease free survival, post-operative morbidity and health economy analysis. RESULTS: By July 2008, 27 hospitals from the Netherlands, Belgium, Germany, Sweden, Spain, Denmark, South Korea and Canada had included 739 patients. The intra-operative conversion rate in the laparoscopic group was 17%. Distribution of age, location of the tumor and radiotherapy were equal in both treatment groups. Most tumors are located in the mid-rectum (41%). CONCLUSION: Laparoscopic surgery in the treatment of rectal cancer is feasible. The results and safety of laparoscopic surgery in the treatment of rectal cancer remain unknown, but are subject of interim analysis within the COLOR II trial. Completion of inclusion is expected by the end of 2009. TRIAL REGISTRATION: Clinicaltrials.gov, identifier: NCT00297791 (www.clinicaltrials.gov).
Asunto(s)
Procedimientos Quirúrgicos del Sistema Digestivo/métodos , Laparoscopía , Neoplasias del Recto/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/cirugía , Selección de Paciente , Proyectos de InvestigaciónRESUMEN
Captopril and mesna are molecules with a free thiol group, used as active ingredients due to their hypotensor and mucolytic properties, respectively. These compounds cross the hematoencephalic barrier and, due to the reactivity of their thiol group, can form adducts with the o-quinones formed during the oxidation of mono- and o-diphenols. Polyphenol oxidase from plants and fungi can be used as a tool for generating o-quinones in their action on o-diphenols and facilitate the formation of adducts in the presence of captopril or mesna. The spectrophotometric characterization of these adducts is useful from several points of view. Here, using the end-point method, which involves the exhaustion of oxygen in the medium, we determined the molar absorptivity of the adducts of different o-diphenols with captopril and mesna. Besides the analytical interest of this approach, we also use it to make a kinetic characterization of polyphenol oxidase as it acts on o-diphenolic substrates that produce unstable o-quinones.
Asunto(s)
Captopril/química , Mesna/química , Fenoles/química , Catecol Oxidasa/metabolismo , Flavonoides/química , Flavonoides/metabolismo , Neurotransmisores/química , Oxidación-Reducción , Fenoles/metabolismo , Polifenoles , Quinonas/química , Espectrofotometría , Compuestos de Sulfhidrilo/químicaRESUMEN
The oxidation of green tea catechins by polyphenol oxidase/O2 and peroxidase/H2O2 gives rise to o-quinones and semiquinones, respectively, which inestability, until now, have hindered the kinetic characterization of enzymatic oxidation of the catechins. To overcome this problem, ascorbic acid (AH2) was used as a coupled reagent, either measuring the disappearance of AH2 or using a chronometric method in which the time necessary for a fixed quantity of AH2 to be consumed was measured. In this way, it was possible to determine the kinetic constants characterizing the action of polyphenol oxidase and peroxidase toward these substrates. From the results obtained, (-) epicatechin was seen to be the best substrate for both enzymes with the OH group of the C ring in the cis position with respect to the B ring. The next best was (+) catechin with the OH group of the C ring in the trans position with respect to the B ring. Epigallocatechin, which should be in first place because of the presence of three vecinal hydroxyls in its structure (B ring), is not because of the steric hindrance resulting from the hydroxyl in the cis position in the C ring. The epicatechin gallate and epigallocatechin gallate are very poor substrates due to the presence of sterified gallic acid in the OH group of the C ring. In addition, the production of H2O2 in the auto-oxidation of the catechins by O2 was seen to be very low for (-) epicatechin and (+) catechin. However, its production from the o-quinones generated by oxidation with periodate was greater, underlining the importance of the evolution of the o-quinones in this process. When the [substrate] 0/[IO4 (-)] 0 ratio = 1 or >>1, H2O2 formation increases in cases of (-) epicatechin and (+) catechin and practically is not affected in cases involving epicatechin gallate, epigallocatechin, or epigallocatechin gallate. Moreover, the antioxidant power is greater for the gallates of green tea, probably because of the greater number of hydroxyl groups in its structure capable of sequestering and neutralizing free radicals. Therefore, we kinetically characterized the action of polyphenol oxidase and peroxidase on green tea catechins. Furthermore, the formation of H2O2 during the auto-oxidation of these compounds and during the evolution of their o-quinones is studied.
Asunto(s)
Catequina/química , Catequina/metabolismo , Té/enzimología , Ácido Ascórbico , Catecol Oxidasa/metabolismo , Indicadores y Reactivos , Cinética , Oxidación-Reducción , Peroxidasa/metabolismo , Té/químicaRESUMEN
Taking as starting point the complete analysis of mean residence times in linear compartmental systems performed by Garcia-Meseguer et al. (Bull. Math. Biol. 65:279-308, 2003) as well as the fact that enzyme systems, in which the interconversions between the different enzyme species involved are of first or pseudofirst order, act as linear compartmental systems, we hereby carry out a complete analysis of the mean lifetime that the enzyme molecules spend as part of the enzyme species, forms, or groups involved in an enzyme reaction mechanism. The formulas to evaluate these times are given as a function of the individual rate constants and the initial concentrations of the involved species at the onset of the reaction. We apply the results to unstable enzyme systems and support the results by using a concrete example of such systems. The practicality of obtaining the mean times and their possible application in a kinetic data analysis is discussed.
Asunto(s)
Estabilidad de Enzimas , Enzimas/química , Modelos Químicos , Algoritmos , Enzimas/metabolismo , Cinética , Modelos LinealesRESUMEN
Tyrosinase is a copper enzyme with broad substrate specifity toward a lot of phenols with different biotechnological applications. The availability of quick and reliable measurement methods of the enzymatic activity of tyrosinase is of outstanding interest. A series of spectrophotometric methods for determining the monophenolase and diphenolase activities of tyrosinase are discussed. The product of both reactions is the o-quinone of the corresponding monophenol/diphenol. According to the stability and properties of the o-quinone, the substrate is classified as four substrate types. For each of these substrate types, we indicate the best method for measuring diphenolase activity (among eight methods) and, when applicable, for measuring monophenolase activity (among four methods). The analytical and numerical solutions to the system of differential equations corresponding to the reaction mechanism of each case confirm the underlying validity of the different spectrophotometric methods proposed for the kinetic characterization of tyrosinase in its action on different substrates.
Asunto(s)
Catecol Oxidasa/análisis , Monofenol Monooxigenasa/análisis , Monofenol Monooxigenasa/metabolismo , Oxidorreductasas/análisis , Espectrofotometría/métodos , Catecol Oxidasa/metabolismo , Cinética , Oxidorreductasas/metabolismo , Especificidad por SustratoRESUMEN
This study explains the action of compounds such as 6-tetrahydrobiopterin, (6BH4) and 6,7-dimethyltetrahydrobiopterin (6,7-di-CH3BH4) on the monophenolase and diphenolase activities of tyrosinase. These reductants basically act by reducing the o-quinones, the reaction products, to o-diphenol. In the case of the diphenolase activity a lag period is observed until the reductant is depleted; then the system reaches the steady-state. In the action of the enzyme on monophenol substrates, when the reductant concentration is less than that of the o-diphenol necessary for the steady-state to be reached, the system undergoes an apparent activation since, in this way, the necessary concentration of o-diphenol will be reached more rapidly. However, when the reductant concentration is greater than that of the o-diphenol necessary for the steady-state to be reached, the lag period lengthens and is followed by a burst, by means of which the excess o-diphenol is consumed, the steady-state thus taking longer to be reached. Moreover, in the present kinetic study, we show that tyrosinase is not inhibited by an excess of monophenol, although, to confirm this, the system must be allowed to pass from the transition state and enter the steady-state, which is attained when a given amount of o-diphenol has accumulated in the medium.
Asunto(s)
Bioquímica/métodos , Monofenol Monooxigenasa/química , Oxidorreductasas/química , Fenoles/química , Pteridinas/química , Química Farmacéutica/métodos , Activación Enzimática , Inhibidores Enzimáticos/química , Enzimas/química , Cinética , Melanocitos/metabolismo , Modelos Químicos , Fenol/química , Espectrofotometría/métodos , Factores de TiempoRESUMEN
Starting from a simple general reaction mechanism of activation of aspartic proteinases zymogens involving a uni- and a bimolecular simultaneous activation route and a reversible inhibition step, the time course equation of the zymogen, inhibitor and activated enzyme concentrations have been derived. Likewise, expressions for the time required for any reaction progress and the corresponding mean activation rates as well as the half-life of the global zymogen activation have been derived. An experimental design and kinetic data analysis is suggested to estimate the kinetic parameters involved in the reaction mechanism proposed.
Asunto(s)
Ácido Aspártico Endopeptidasas/química , Inhibidores Enzimáticos/química , Precursores Enzimáticos/química , Modelos Químicos , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Catálisis , Activación Enzimática , Precursores Enzimáticos/antagonistas & inhibidores , CinéticaRESUMEN
Chlorogenic acid is the major diphenol of many fruits, where it is oxidized enzymatically by polyphenol oxidase (PPO) or peroxidase (POD) to its o-quinone. In spectrophotometric studies of chlorogenic acid oxidation with a periodate ratio of [CGA]0/[IO4-]0 < 1 and [CGA]0/[IO4-]0 > 1, the o-quinone was characterized as follows: lambda(max) at 400 nm and epsilon = 2000 and 2200 M-1 cm-1 at pH 4.5 and 7.0, respectively. In studies of o-quinone generated by the oxidation of chlorogenic acid using a periodate at ratio of [CGA]0/[IO4-]0 > 1, a reaction with the remaining substrate was detected, showing rate constants of k = 2.73 +/- 0.17 M-1 s-1 and k' = 0.05 +/- 0.01 M-1 s-1 at the above pH values. A chronometric spectrophotometric method is proposed to kinetically characterize the action of the PPO or POD on the basis of measuring the time it takes for a given amount of ascorbic acid to be consumed in the reaction with the o-quinone. The kinetic constants of mushroom PPO and horseradish POD are determined.
Asunto(s)
Catecol Oxidasa/metabolismo , Ácido Clorogénico/metabolismo , Peroxidasa/metabolismo , Quinonas/metabolismo , Agaricales/enzimología , Armoracia/enzimología , Concentración de Iones de Hidrógeno , Cinética , Oxidación-Reducción , Ácido Peryódico/metabolismoRESUMEN
This paper presents the derivation, under a minimal set of assumptions, of a general expression for the steady-state fractional modification of an interconvertible protein involved in four different schemes of monocyclic enzyme cascade systems. From this general expression we derive, as particular cases, other, simpler expressions by applying additional assumptions and which have, therefore, a smaller range of validity. Some of these particular expressions coincide with those already obtained in previous contributions on individualised analyses. We discuss the relationships between the kinetic parameters and the concentrations needed for the fulfilment of the additional assumptions. The goodness of the analysis was tested by reference to the shape in the steady-state of the simulated time progress curves obtained by numerical integration.
Asunto(s)
Enzimas/química , Modelos Químicos , Procesamiento Proteico-Postraduccional , Algoritmos , Regulación Alostérica , Activación Enzimática , Cinética , Reproducibilidad de los ResultadosRESUMEN
Starting from a simple general reaction mechanism of activation of aspartic proteinase zymogens involving an uni- and a bimolecular simultaneous route, the time course equation of the concentration of the zymogen and of the activated enzyme have been derived. From these equations, an analysis quantifying the relative contribution to the global process of the two routes has been carried out for the first time. This analysis suggests a way to predict the time course of the relative contribution as well as the effect of the initial zymogen and activating enzyme concentrations, on the relative weight. An experimental design and kinetic data analysis is suggested to estimate the kinetic parameters involved in the reaction mechanism proposed. Finally, we apply some of our results to experimental data obtained by other authors in experimental studies of the activation of some aspartic proteinase zymogens.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Simulación por Computador , Precursores Enzimáticos/metabolismo , Modelos Químicos , Vesículas Secretoras/metabolismo , Animales , Activación Enzimática , Cinética , Modelos BiológicosRESUMEN
The molar absorptivities of the quinones produced from different o-diphenols, triphenols, and flavonoids were calculated by generating the respective quinones through oxidation with an excess of periodate. Oxidation of these substrates by this reagent was analogous to oxidation by tyrosinase with molecular oxygen, although the procedure showed several advantages over the enzymatic method in that oxidation took place almost immediately and quinone stability was favored because no substrate remained. The o-diphenols studied were pyrocatechol, 4-methylcatechol, 4-tert-butylcatechol, 3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenylethylamine, 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxyphenylpropionic acid, and caffeic acid; the triphenols studied were pyrogallol, 1,2,4-benzenetriol, 6-hydroxydopa, and 6-hydroxydopamine; and the flavonoids studied were (+)catechin, (-)epicatechin, and quercetin. In addition, the stability of the quinones generated by oxidation of the compounds by [periodate]0/[substrate]0 << 1 was studied. Taking the findings into account, tyrosinase could be measured by following o-quinone formation in rapid kinetic studies using the stopped-flow method. However, measuring o-quinone formation could not be useful for steady-state studies. Therefore, several methods for following tyrosinase activity are proposed, and a kinetic characterization of the enzyme's action on these substrates is made.
Asunto(s)
Benzoquinonas/química , Monofenol Monooxigenasa/metabolismo , Cinética , Oxidación-Reducción , Espectrofotometría Ultravioleta , Especificidad por SustratoRESUMEN
The time course of the residual enzyme activity of a general model consisting of an autocatalytic zymogen activation process inhibited by an irreversible competitive inhibitor and an irreversible uncompetitive inhibitor has been studied. Approached analytical expressions which furnish the time course of the residual enzyme activity from the onset of the reaction depending on the rate constants and initial concentration have been obtained. The goodness and limitations of the analytical equations were checked by comparing with the results obtained from the numerical integration, i.e. with the simulated progress curves. A dimensionless parameter giving the relative contributions of both the activation and the inhibitions routes is suggested, so that the value of this parameter determines whether the activation or the inhibitions routes prevail or if both processes are balanced during the time for which the analytical expressions are valid. The effects of the initial zymogen, free enzyme and inhibitors concentrations are analysed. Finally an experimental design and kinetic data analysis is proposed to evaluate simultaneously the kinetic parameters involved and to discriminate between different zymogen activation processes which can be considered particular cases of the general model.
Asunto(s)
Inhibidores Enzimáticos/química , Precursores Enzimáticos/metabolismo , Unión Competitiva , Catálisis , Simulación por Computador , Inhibidores Enzimáticos/metabolismo , CinéticaRESUMEN
Thiols, such as cysteine and N-acetylcysteine, are included in many pharmaceutical products for their mucolytic properties. The method described here uses mushroom polyphenol oxidase (PPO) to determine two thiols and consists of measuring the lag period in the formation of the product generated as PPO acts on o-diphenol in the presence of a thiol. In the experimental conditions, o-quinone is formed enzymatically and then reacts stoichiometrically with the thiol, originating the corresponding thiol-diphenol adduct, which does not absorb visible light. Once the thiol has been used up, the o-quinone can be observed in the medium. It must be borne in mind that the inhibition of PPO is practically null at low concentrations of thiol, and the only effect observed is the formation of the thiol-diphenol adduct. In the following, an exact kinetic method capable of rapidly and accurately assaying thiols with PPO and o-diphenol is optimized and is shown to be a straightforward way of calculating thiol concentration. The method has been successfully applied to the determination of cysteine in model solutions and of N-acetylcysteine in pharmaceutical products.
Asunto(s)
Acetilcisteína/análisis , Catecol Oxidasa , Cisteína/análisis , Espectrofotometría , Agaricales/enzimología , Catecol Oxidasa/metabolismo , Cinética , Espectroscopía de Resonancia Magnética , Compuestos de Sulfhidrilo/análisis , Compuestos de Sulfhidrilo/metabolismoAsunto(s)
Endotelio Vascular/enzimología , Hipertensión/enzimología , Óxido Nítrico Sintasa/genética , Óxido Nítrico/genética , Estudios de Casos y Controles , Endotelio Vascular/fisiopatología , Factores Relajantes Endotelio-Dependientes/análisis , Femenino , Humanos , Hipertensión/fisiopatología , Masculino , Persona de Mediana Edad , Óxido Nítrico/análisis , Óxido Nítrico Sintasa/análisis , Óxido Nítrico Sintasa de Tipo IIIRESUMEN
In order to unify and generalize, we define the International Units used to express the monophenolase and diphenolase activity of mushroom tyrosinase acting on different monophenol/diphenol pairs and establish a quantitative relation. Similarly, the activity units to express tyrosinase activity proposed by suppliers are discussed and compared with the above International Units. Lastly, we study the relation between International Units of diphenolase activity and of monophenolase activity for other biological sources of tyrosinase.