Purification and characterization of a new trypsin inhibitor from Dimorphandra mollis seeds.
A second trypsin inhibitor (DMTI-II) was purified from the seed of Dimorphandra mollis (Leguminosae-Mimosoideae) by ammonium sulfate precipitation (30-60%), gel filtration, and ion-exchange and affinity chromatography. A molecular weight of 23 kDa was estimated by gel filtration on a Superdex 75 column SDS-PAGE under reduced conditions showed that DMTI-II consisted of a single polypeptide chain, although isoelectric focusing revealed the presence of three isoforms. The dissociation constant of 1.7 x 10(-9) M with bovine trypsin indicated a high affinity between the inhibitor and this enzyme. The inhibitory activity was stable over a wide pH range and in the presence of DTT. The N-terminal sequence of DMTI-II showed a high degree of homology with other Kunitz-type inhibitors.
Mello
G C
GC
Departamento de Bioquimica, Instituto de Biologia, Universidade Estadual de Campinas, SP, Brazil.
Oliva
M L
ML
Sumikawa
J T
JT
Machado
O L
OL
Marangoni
S
S
Novello
J C
JC
Macedo
M L
ML
D000818:Animals; D004229:Dithiothreitol / Q000378:metabolism; D007887:Fabaceae / Q000737:chemistry*; D006801:Humans; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012639:Seeds / Q000737:chemistry*; D013379:Substrate Specificity; D013696:Temperature; D014357:Trypsin / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D010940:Plant Proteins; D014361:Trypsin Inhibitors; D014357:Trypsin; D004229:Dithiothreitol