Hidrolytic Profile and isolation of the Proteolytic Components of Latex from Araujia angustifolia Fruits

OBREGÓN, WALTER; CURCIARELLO, RENATA; CAFFINI, NÉSTOR; PRIOLO, NORA

ACTA FARM. BONAERENSE

Colegio de Farmacéuticos de la Provincia de Buenos Aires

Lugar: La Plata; Año: 2006 vol. 25 p. 206 - 212

0326-2383

The presence of hydrolases in the latex of Araujia angustifolia (Asclepiadaceae), a climbing plant that grows in Argentina, has been studied. The crude extract (CE) obtained by differential centrifugation at 8000 and 15000 rpm of latex from A. angustifolia fruits, collected on 0.05 M citric-citrate buffer (pH 4.5) with 5 mM EDTA, exhibited several enzymatic activities. CE showed amydasic activity on L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA), proteolytic activity on 1% casein in Tris-HCl pH 8.5, polygalacturonidase activity using 1 % polygalacturonic acid in acetic acetate buffer pH 4.5, pectin methyl esterase on 1% pectin in citric phosphate pH 6.8 in the presence of 0.02M Cl2Ca, and endosterolytic activity on p-nitrophenyl esters of N-a-carbobenzoxy-L-amino acids in 0.1 M Tris-HCl buffer pH 8.0. As proteolytic activity was the main hydrolytic activity observed, the proteolytic properties of CE were stablished. The proteases present in CE showed the characteristics of the cysteine proteases: optimum pH at alkaline range, isoelectric point (pI) higher than 8.0, and inhibition of activity by thiol blocking reagents, such as E-64 and iodoacetate. A remarkable thermal stability was also evident in the CE. Three proteases have been detected by IEF and zymogram in the CE and purified by FPLC affording three basic active fractions (araujiain aI, aII and aIII) with molecular masses about 23 kDa (SDS-PAGE).