Isolation, characterization and analysis of the agglutinative activity of a lectin from Crotalaria spectabilis
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Lectins are proteins with ability to recognize specific carbohydrates. These are present in virtually all organisms and have increasing applications in biotechnology. Here, our aim was to purify lectins from seeds of Crotalaria spectabilis Roth and determine their agglutinative ability. In this study, 45 g of seeds were milled, their proteins were precipitated by acetone or ammonium sulfate and purified by exclusion and ion-exchange chromatography. An isolated lectin was submitted to tests for hemagglutination and inhibition of hemagglutinating activity by carbohydrates as well as tests for its response to chelating and reducing agents. Our results show that the apparent molecular weight (as determined by SDS-PAGE) of the lectin is 30 kDa, and the tests for inhibition of erythrocytes’ agglutinative activity by sugars were positive for d-galactose and N-acetyl-d-galactosamine. Data obtained with the chelating agent EDTA demonstrated the presence of divalent cations in the protein structure. However, the reducing agent 2-mercaptoethanol was unable to inhibit the protein’s bioactivity. The lectin agglutinated the blood groups A, B, AB and O, as well as bacterial lineages from the species Leptospira interrogans and Leptospira biflexa, indicating a prospective application in the diagnosis and treatment of leptospirosis.
KeywordsCrotalaria spectabilis Lectin Purification Hemagglutination Leptospirosis
The authors would like to thank to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the financial support, to the Agronomic Institute of Campinas (IAC), to the Laboratory of Genetics from Bahia State University, and to the collaboration of the Laboratories from Federal University of Bahia (UFBA): Laboratory of Bioprospection and Biotechnology (LABBIOTEC), Laboratory of Research in Materia Medica (LAPEMM), Laboratory of Genetics and Plant Evolution (LAGEV), Laboratory of Human Genetics and Mutagenesis (LGHM), and the Group of Research in Chemical Synthesis and Molecular Biodiversity (GPSQ). The authors also would like to thank Dr. Eduardo Ximenes (University of Perdue, USA) for revising the manuscript.
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Conflict of interest
The authors declare that they have no conflicts of interest.
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