Abstract
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.
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Abbreviations
- BAPNA:
-
N-α-benzoyl-dl- arginyl-p-nitroanilide
- BPTI:
-
Bovine pâncreas trypsin inhibitor
- BSA:
-
Bovine serum albumin
- BTPNA:
-
N-benzoyl-l-tyrosine-p-nitroanilide
- CHD:
-
1,2-cyclohexanedione
- CNBr:
-
Cyanogen bromide
- DTT:
-
Dithiothreitol
- HEPES:
-
[4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid]
- HNE:
-
Human neutrophil elastase
- PTC:
-
Phenylisothiocyanate
- SBTI:
-
Soybean trypsin inhibitor
- TFA:
-
Acetonitrilie and trifluoroacetic acid
- TLCK:
-
Tosyl-l-lysine chloromethyl ketone
- TNBS:
-
Trinitrobenzenesulphonic acid
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Acknowledgments
This work was supported by FUNDECT (Fundacão de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), and PROPP/UFMS (Pró-Reitoria de Pesquisa e Pós-graduação da Universidade Federal de Mato Grosso do Sul).
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Macedo, M.L.R., Diz Filho, E.B.S., Freire, M.G.M. et al. A Trypsin Inhibitor from Sapindus saponaria L. Seeds: Purification, Characterization, and Activity Towards Pest Insect Digestive Enzyme. Protein J 30, 9–19 (2011). https://doi.org/10.1007/s10930-010-9296-7
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DOI: https://doi.org/10.1007/s10930-010-9296-7