Isolation of a trypsin inhibitor from Echinodorus paniculatus seeds by affinity chromatography on immobilized Cratylia mollis isolectins.
A highly purified trypsin inhibitor was obtained from Echinodorus paniculatus when an extract prepared from E. paniculatus seed flour (25 gl(-1), with 0.1 M ammonium acetate buffer, pH 8.3, under agitation for 6 min at 28 degrees C) was chromatographed on Sephadex G-25 (12 mlh(-1)), followed by affinity chromatography on immobilized Cratylia mollis isolectins (Cra Iso 1,2,3-Sepharose). The column chromatography was performed at 24 degrees C; the matrix was washed (30 mlh(-1)) with 0.1 M sodium phosphate buffer, pH 7.4 or with the same buffer containing 0.2 M glucose, followed by application of inhibitor sample and elution with 0.015 M sodium borate buffer, pH 7.4, or 1.0 M NaCl. A purified fraction of inhibitor was obtained by gel filtration chromatography (GF-450/HPLC column). Trypsin inhibitory activity was eliminated when the inhibitor was treated with metaperiodate showing that the carbohydrate moiety was important for trypsin inhibition. Binding of inhibitor was also evaluated on immobilized concanavalin A (Con A-Sepharose) using previously described chromatographic conditions with results similar to Cra Iso 1,2,3-Sepharose chromatography.
Paiva
P M G
PM
Departamento de Bioquímica, CBB/UFPE, Av. Moraes Rego, S/N, Cidade Universitária, Recife-PE, CEP 50670-420, Brazil. pmgpaiva@bol.com.br
Souza
A F
AF
Oliva
M L V
ML
Kennedy
J F
JF
Cavalcanti
M S M
MS
Coelho
L C B B
LC
Sampaio
C A M
CA
D034901:Alismataceae / Q000737:chemistry*; D002846:Chromatography, Affinity; D004791:Enzyme Inhibitors / Q000302:isolation & purification*; D007887:Fabaceae / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D037102:Lectins / Q000737:chemistry; D010940:Plant Proteins / Q000302:isolation & purification*; D012639:Seeds / Q000737:chemistry; D014361:Trypsin Inhibitors; D000516:alpha-Amylases / Q000037:antagonists & inhibitors
D004791:Enzyme Inhibitors; D037102:Lectins; D010940:Plant Proteins; D014361:Trypsin Inhibitors; D000516:alpha-Amylases