A trypsin inhibitor from Sapindus saponaria L. seeds: purification, characterization, and activity towards pest insect digestive enzyme.
The present paper describes the purification, characterization and determination of the partial primary structure of the first trypsin inhibitor isolated from the family Sapindaceae. A highly stable, potent trypsin inhibitor (SSTI) was purified to homogeneity. SDS-PAGE analysis revealed that the protein consists of a two-polypeptide chain with molecular masses of approximately 15 and 3 kDa. The purified inhibitor inhibited bovine trypsin at a 1:1 M ratio. Kinetic analysis revealed that the protein is a competitive inhibitor with an equilibrium dissociation constant of 10⁻⁹ M for trypsin. The partial NH₂- terminal sequence of 36 amino acids in SSTI indicates homology with other members of the trypsin-inhibitor family from different sources. This inhibitor is highly stable in the presence of denaturing agents. SSTI showed significant inhibitory activity against trypsin-like proteases present in the larval midgut on Anagasta kuehniella, Corcyra cephalonica, Diatreae saccharalis and Anticarsia gemmatalis.
Laboratório de Purificação de Proteínas e suas Funções Biológicas, Departamento de Tecnologia de Alimentos e Saúde Pública, Centro de Ciências Biológicas e da Saúde, Universidade Federal do Mato Grosso do Sul (UFMS), Cidade Universitária S/N, Caixa Postal 549, Campo Grande, MS, CEP 79070-900, Brazil. bioplant@terra.com.br
Maria Lígia R
ML
Macedo
Eduardo B S
EB
Diz Filho
Mariadas Graças M
MG
Freire
Maria Luiza V
ML
Oliva
Joana T
JT
Sumikawa
Marcos H
MH
Toyama
Sérgio
S
Marangoni
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001938:Brazil; D004229:Dithiothreitol / Q000737:chemistry / Q000494:pharmacology; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D007306:Insecticides / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D007421:Intestine, Small / Q000201:enzymology; D007700:Kinetics; D007814:Larva / Q000201:enzymology; D007915:Lepidoptera; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D055550:Protein Stability; D032127:Sapindus / Q000737:chemistry*; D012639:Seeds / Q000737:chemistry*; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology
D007306:Insecticides; D010940:Plant Proteins; D014361:Trypsin Inhibitors; D001120:Arginine; D004229:Dithiothreitol