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A Trypsin Inhibitor from Sapindus saponaria L. Seeds: Purification, Characterization, and Activity Towards Pest Insect Digestive Enzyme

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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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Authors and Affiliations

  1. 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

    Maria Lígia R. Macedo

  2. Departamento de Bioquímica, Instituto de Biologia, Unicamp, Campinas, SP, Brazil

    Eduardo B. S. Diz Filho, Marcos H. Toyama & Sérgio Marangoni

  3. Laboratório de Química e Biomoléculas, Centro de Pesquisa, Institutos Superiores do CENSA, Campos dos Goytacazes, RJ, Brazil

    Mariadas Graças M. Freire

  4. Departamento de Bioquímica, Escola Paulista de Medicina, UNIFESP, São Paulo, Brazil

    Maria Luiza V. Oliva & Joana T. Sumikawa

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  1. Maria Lígia R. Macedo
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Correspondence to Maria Lígia R. Macedo.

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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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