RESUMEN
In a previous study, we showed that purified commercial esterase activity can be detected in a chemiluminescent assay based on the hydrolysis of 2-methyl-1-propenylbenzoate (MPB) to 2-methyl-1-propenol, which is subsequently oxidized by the horseradish peroxidase (HRP)-H(2)O(2) system. The purpose of this study was to verify the applicability of this assay to human serum. The existence of an esterase activity capable of hydrolysing MPB is indicated by the fact that the MPB-serum-HRP-H(2)O(2) system consumes oxygen and emits light. Both signals were abolished by prior serum heat inactivation and were preserved when serum was stored at < or =4 degrees C. Addition of aliesterase inhibitors, such as fluoride ion and trichlorfon or the cholinesterase inhibitor eserine, totally prevents light emission. The butyrylcholinesterase-specific substrate benzoylcholine causes a delay in both O(2) uptake and light emission, while the specific acetylcholinesterase substrate, acetyl-beta-methylcholine, had practically no effect. Purified butyrylcholinesterase, but not acetylcholinesterase, triggered light emission. The finding that butyrylcholinesterase is responsible for the hydrolysis of MPB in serum should serve as the basis for the development of a specific chemiluminescent assay for this enzyme.
Asunto(s)
Benzoatos/química , Butanoles/química , Butirilcolinesterasa/sangre , Inhibidores de la Colinesterasa/química , Animales , Benzoatos/metabolismo , Benzoilcolina/química , Benzoilcolina/metabolismo , Butanoles/metabolismo , Bovinos , Inhibidores de la Colinesterasa/metabolismo , Eritrocitos/enzimología , Fluoruros/química , Peroxidasa de Rábano Silvestre/química , Peroxidasa de Rábano Silvestre/metabolismo , Humanos , Hidrólisis , Mediciones Luminiscentes , Cloruro de Metacolina/química , Cloruro de Metacolina/metabolismo , Fisostigmina/química , Fisostigmina/metabolismo , Triclorfón/químicaRESUMEN
Esterase from monocytes promotes the hydrolysis of 2-methyl-1-propenylbenzoate (MPB) yielding 2-methyl-1-propenol, which is oxidized by horseradish peroxidase/H2O2 producing triplet acetone. The chemiluminescence of this reaction can be enhanced by the addition of 9,10-dibromoanthracene-2-sulphonate. The non-specific esterase present in monocytes is responsible for MPB hydrolysis, since (a) the chemiluminescence of the reaction was inhibited by fluoride, and (b) cells that do not contain a significant amount of non-specific esterases, e.g. lymphocytes and neutrophils, did not trigger light emission. The analytical application of this reaction is considered.
Asunto(s)
Esterasas/sangre , Granulocitos/enzimología , Linfocitos/enzimología , Monocitos/enzimología , Benzoatos , Colorantes Fluorescentes , Peroxidasa de Rábano Silvestre , Humanos , Peróxido de Hidrógeno , Indicadores y Reactivos , Cinética , Mediciones LuminiscentesRESUMEN
The hydrolysis of 2-methyl-1-propenylbenzoate catalyzed by esterase produces 2-methyl-1-propenol, which can be subsequently oxidized by the H2O2/horseradish peroxidase (HRP) system to yield electronically excited triplet acetone. The level of luminescence elicited by this species is proportional to total esterase used, making it possible to determine as little as 2 pmol of esterase. Yet, its intensity can be enhanced several orders of magnitude by fluorescent acceptors like sodium 9,10-dibromoanthracene-2-sulfonate. The system works as a chemiluminescent reaction triggered by esterase and can be used to elaborate analytical assays to determine its activity. This chemiluminescence is also promoted by HRP conjugates instead of free HRP and, hence, this simple reaction system can also be used to develop sensitive chemiluminescent immunoassays based upon peroxidase activity.