RESUMEN
Hypertension and related diseases afflict millions of individuals worldwide, and many investigations of angiotensin I-converting enzyme (ACE) activity have been carried out. Most of these have used hippuryl-histidyl-leucine (HHL) as a substrate for ACE reaction with considerable interferences. Here we propose the use of a new substrate, 3-hydroxybutyrylglycyl-glycyl-glycine (3HB-GGG) for the screening of ACE inhibitors. Under the actions of ACE and aminoacylase, 3HB-GGG is cleaved into amino acids (Gly and Gly-Gly) and 3-hydroxybutyric acid (3HB). The assay conditions were optimized and applied to monitor the ACE inhibitory activity in terms of 3HB measured using an F-kit. Under the optimum assay parameters-ACE (0.2 U/ml) and aminoacylase (172 kU/ml) incubated with 3HB-GGG (3.4 mg/ml) at 37 degrees C for 30 min-the Gly-Gly and Gly cleaved from 3HB-GGG by enzymes was able to be identified, affirming the feasibility of substituting 3HB-GGG for the conventional substrate HHL. In addition, the current method was more sensitive, accurate, rapid, and convenient than the conventional method.
Asunto(s)
Ácido 3-Hidroxibutírico/análisis , Ácido 3-Hidroxibutírico/química , Inhibidores de la Enzima Convertidora de Angiotensina/química , Inhibidores de la Enzima Convertidora de Angiotensina/clasificación , Hidroxibutiratos , Oligopéptidos/análisis , Oligopéptidos/química , Ácido 3-Hidroxibutírico/síntesis química , Animales , Bioquímica/métodos , Calibración , Captopril/química , Evaluación Preclínica de Medicamentos/métodos , Fagopyrum/química , Estudios de Factibilidad , Análisis de los Alimentos/métodos , Formazáns/química , Ajo/química , Hipuratos/química , Hidrólisis , Indicadores y Reactivos , Concentración 50 Inhibidora , Estructura Molecular , Oligopéptidos/síntesis química , Conejos , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrofotometría , Especificidad por SustratoRESUMEN
The enzyme xanthine oxidase (XOD) has been recognized as a key enzyme causing oxidative injury to tissues by ischemia-reperfusion. For this reason, XOD inhibitor, which effectively suppresses this enzyme, plays an important role in the inhibition of many diseases related to reactive oxygen species (ROS). In order to screen XOD inhibitors rapidly and conveniently, a novel assay using flow injection analysis (FIA) was proposed in the present investigation. To optimize the practical FIA system, we studied the effect of the reagent concentrations and the flow condition on the enzymatic reaction, and then selected the optimum condition as follows: 200-mU/ml XOD concentration, 0.5-mM xanthine concentration, 0.5-ml/min flow rate, and 2-m mixing coil length. Under this condition, a typical XOD inhibitor quercetin was determined in the concentration range 0.1 - 1.5 mM at a sampling frequency of 10 samples/h. Using the optimized FIA method, we determined the XOD inhibitory activity of some food samples: onions, apples and teas, which are the high sources of flavonoids known as the potential XOD inhibitors. Among these samples, tea leaves showed the highest activity, the second was onions and the lowest was apples. Based on the result of the assay, not only quercetin, but also other components in investigated samples, contributed to the XOD inhibitory activity.