RESUMO
INTRODUCCIÓN: la mayor parte de la producción mundial de enzimas está destinada a la obtención de proteasas. Las aplicaciones de estas biomoléculas con fines terapéuticos son muy variadas. Algunas se conocen como remedios naturales y otras son fármacos modificadores de la respuesta biológica. Las plantas de piña contienen varias cisteíno-proteasas, el componente mayoritario aislado del tallo es la bromelina de tallo (EC 3.4.22.32). En el campo de la salud se le atribuyen varias acciones entre las que se destaca la de inducir la diferenciación de células tumorales y atenuar el crecimiento del tumor. OBJETIVOS: caracterizar cinéticamente un preparado semipurificado de bromelina obtenida por un procedimiento desarrollado en Cuba para ser evaluado en el campo de la salud. MÉTODOS: el extracto crudo de bromelina, obtenido a partir de tallos de plantas adultas de Ananas comosus (L.) Merr cv Española roja (piña) se purificó por cromatografía de intercambio iónico en carboximetilcelulosa-52 en un sistema semibach. RESULTADOS: la caracterización cinética del extracto purificado permitió comprobar la existencia de un preparado activo y estable con una actividad específica de 0,94 U/mg de proteínas (2,34 veces superior a la del extracto crudo original), con una fracción proteica mayoritaria de masa molar 24 500 Da y un punto isoeléctrico, localizado en la zona de pH básico (9,55). Tiene pH óptimo 6,8 al utilizar hemoglobina 2 por ciento como sustrato a 37 ºC y una estabilidad en función del pH y de la temperatura aceptable. Si se conserva liofilizado a - 20 ºC mantiene 80 por ciento de la actividad durante 1 año. CONCLUSIONES: la cromatografía de intercambio iónico en carboximetilcelulosa-52 posibilitó la obtención de un preparado semipurificado de bromelina, con una actividad específica superior a la del extracto original que puede ser utilizado en la medicina como antitumoral
INTRODUCTION: most of the world production of enzymes is devoted to proteases obtaining. Applications of these biomolecules for therapeutical objectives are very varied. Some are known as natural remedies and other are drugs modifying the biological response. Pineapple plants contain some cysteine-proteases, the majority component isolated from stem is the stem Bromelain (EC 3.4.22.32). In the health field it has attributed some actions including the differentiation induction of tumor cells and attenuation of tumor growth. OBJECTIVES: to characterize kinetically a semipurified preparation from Bromelain obtained by a procedure developed in Cuba to be assessed in the health field. METHODS: the crude extract from Bromelain obtained from the adult plant stems of Ananas comosus (L.) Merr Cv Spanish red (pineapple) was purified by ion-exchange chromatography in carboxylmethylcelulose-52 in semibatch system. RESULTS: kinetic characterization of purified extract allowed us to verify the existence of a stable and active preparation with a specific activity of 0,94 U/mg of proteins (2,34 times superior to that of original crude extract) with a protein fraction in the main of molar mass 24 500 Da and a isoelectric point, located in the basic pH zone (9,55). It has a 6,8optimal pH using the 2 percent hemoglobin as substrate at 37ºC and stability depending on pH and on acceptable temperature. If it is preserved lyophilized at 20 ºC it maintains the 80 percent of activity for a year. CONCLUSIONS: the ion-exchange chromatography in carboxyl methylcellulose-52 allowed the achievement of semipurified preparation from Bromelain with a specific activity higher to that of original extract that may to be used in medicine as antitumor agent
Assuntos
Ananas/química , Bromelaínas/uso terapêutico , Neoplasias/tratamento farmacológicoRESUMO
An affinity matrix containing the antimalarial drug target Plm II (plasmepsin II) as ligand was generated. This enzyme belongs to the family of Plasmodium (malarial parasite) aspartic proteinases, known as Plms (plasmepsins). The procedure established to obtain the support has two steps: the immobilization of the recombinant proenzyme of Plm II to NHS (N-hydroxysuccinimide)-activated Sepharose and the activation of the immobilized enzyme by incubation at pH 4.4 and 37 degrees C. The coupling reaction resulted in a high percentage immobilization (95.5%), and the matrices obtained had an average of 4.3 mg of protein/ml of gel. The activated matrices, but not the inactive ones, were able to hydrolyse two different chromogenic peptide substrates and haemoglobin. This ability was completely blocked by the addition of the general aspartic-proteinase inhibitor, pepstatin A, to the reaction mixture. The matrices were useful in the affinity purification of the Plm II inhibitory activity detected in marine invertebrates, such as Xestospongia muta (giant barrel sponge) and the gorgonian (sea-fan coral) Plexaura homomalla (black sea rod), with increases of 10.2- and 5.9-fold in the specific inhibitory activity respectively. The preliminary K(i) values obtained, 46.4 nM (X. muta) and 1.9 nM (P. homomalla), and the concave shapes of the inhibition curves reveal that molecules are reversible tight-binding inhibitors of Plm II. These results validated the use of the affinity matrix for the purification of Plm II inhibitors from complex mixtures and established the presence of Plm II inhibitors in some marine invertebrates.