RESUMEN
We successfully prepared the Se-containing abzyme (Se-abzyme) with glutathione peroxidase (GPX) activity and further studied its physicochemical and enzymic properties and stabilities. Data showed that the isoelectric point of the abzyme was 6.95-7.08, and its molecular weight was 158 KD. The ranges of optimum pH and temperature of the Se-abzyme were wider than the native GPX. The store stability of the abzyme was higher than the native GPX. The Se content in the abzyme was found to be 5 mol Se/mol abzyme by X-ray photoelectron spectrum, and binding constant 1.11 x 10(7)M-1 by using ELISA method. The Se-abzyme was inhibited competitively by dithiobis(2-nitrobenzoic acid) (DTNB), and inhibition constant was determined to be 1.25 x 10(-3)M-1.
Asunto(s)
Enzimas/química , Enzimas/metabolismo , Glutatión Peroxidasa/química , Selenio/análisis , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Glutatión/metabolismo , Glutatión Peroxidasa/antagonistas & inhibidores , Glutatión Peroxidasa/metabolismo , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Peso Molecular , Mutación , Especificidad por Sustrato , TemperaturaRESUMEN
A new strategy for generating abzyme was developed. Glutathione peroxidase (GPX, EC 1.11.1.9) is one of the important members of antioxidation enzyme system; it catalyzes the reductions of a variety of hydroperoxides in presence of glutathione(GSH). We have first prepared the monoclonal antibody (McAb) with GSH binding sites, then incorporated GPX catalytic group selenocystein (SeCys) into the antibody combining sites by using chemical mutation. Thus the mutated antibody displays high GPX activity, which approaches the magnitude level of native GPX, exhibits the kinetic behavior similar to native GPX, and has some advantages over native GPX.
Asunto(s)
Glutatión Peroxidasa/metabolismo , Mutagénesis , Selenio , Animales , Anticuerpos Monoclonales/metabolismo , Sitios de Unión de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Glutatión/análogos & derivados , Glutatión/síntesis química , Glutatión/metabolismo , Glutatión Peroxidasa/análisis , Glutatión Peroxidasa/biosíntesis , Haptenos , Inmunoglobulina G/metabolismo , Cinética , Ratones , Ratones Endogámicos BALB C/inmunología , Conejos/inmunología , Selenocisteína/metabolismoRESUMEN
From Oxytropis ochrocephala Bunge, four saponins 1, 2, 3, and 4 were isolated. Saponins 1 and 2 are new compounds. The common genuine aglycone of the saponins is soyasapogenol B. The structures of those saponins were established by means of 13C-NMR, MS analyses, and chemical methods. In addition, a new interesting artifact aglycone 6 was obtained from the hydrolysate of these saponins and its structure was elucidated by spectral analyses and chemical transformations.