title: Primary Structure of a Trypsin Inhibitor (Copaifera langsdorffii Trypsin Inhibitor-1) Obtained from C. langsdorffii Seeds.
abstract: In this study, the aim was to determine the complete sequence of the Copaifera langsdorffii trypsin inhibitor (CTI)-1 using 2-dimensional (2D)-PAGE, matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF), and quadrupole time-of-flight (QTOF) spectrometry. Spots A (CTI-1) and F (CTI-2) were submitted to enzymatic digestions with trypsin, SV8, and clostripain. The accurate mass of the peptide obtained from each digest was determined by mass spectrometry (MS) using MALDI-TOF. The most abundant peptides were purified and sequenced in a liquid chromatograph connected to an electrospray ionization-QTOF MS. When the purified trypsin inhibitor was submitted to 2D electrophoresis, different spots were observed, suggesting that the protein is composed of 2 subunits with microheterogeneity. Isoelectric points of 8.0, 8.5, and 9.0 were determined for the 11 kDa subunit and of 4.7, 4.6, and 4.3 for the 9 kDa subunit. The primary structure of CTI-1, determined from the mass of the peptide of the enzymatic digestions and the sequence obtained by MS, indicated 180 shared amino acid residues and a high degree of similarity with other Kunitz (KTI)-type inhibitors. The peptide also contained an Arg residue at the reactive site position. Its 3-dimensional structure revealed that this is because the structural discrepancies do not affect the canonical conformation of the reactive loop of the peptide. Results demonstrate that a detailed investigation of the structural particularities of CTI-1 could provide a better understanding of the mechanism of action of these proteins, as well as clarify its biologic function in the seeds. CTI-1 belongs to the KTI family and is composed of 2 polypeptide chains and only 1 disulfide bridge.
authors.affiliation: 1 Campus Centro Oeste, Federal University of São João Del Rei, Divinópolis, Brazil; and 2 Departamento de Bioquímica and 3 Departamento de Química, Universidade Estadual de Campinas, Campinas, Brazil.
authors.forename: José A
authors.initials: JA
authors.lastname: Silva
authors.affiliation: 1 Campus Centro Oeste, Federal University of São João Del Rei, Divinópolis, Brazil; and 2 Departamento de Bioquímica and 3 Departamento de Química, Universidade Estadual de Campinas, Campinas, Brazil.
authors.forename: Dávia G
authors.initials: DG
authors.lastname: Pompeu
authors.affiliation: 1 Campus Centro Oeste, Federal University of São João Del Rei, Divinópolis, Brazil; and 2 Departamento de Bioquímica and 3 Departamento de Química, Universidade Estadual de Campinas, Campinas, Brazil.
authors.forename: Marcus B
authors.initials: MB
authors.lastname: Smolka
authors.affiliation: 1 Campus Centro Oeste, Federal University of São João Del Rei, Divinópolis, Brazil; and 2 Departamento de Bioquímica and 3 Departamento de Química, Universidade Estadual de Campinas, Campinas, Brazil.
authors.forename: Fabio C
authors.initials: FC
authors.lastname: Gozzo
authors.affiliation: 1 Campus Centro Oeste, Federal University of São João Del Rei, Divinópolis, Brazil; and 2 Departamento de Bioquímica and 3 Departamento de Química, Universidade Estadual de Campinas, Campinas, Brazil.
authors.forename: Moacyr
authors.initials: M
authors.lastname: Comar
authors.affiliation: 1 Campus Centro Oeste, Federal University of São João Del Rei, Divinópolis, Brazil; and 2 Departamento de Bioquímica and 3 Departamento de Química, Universidade Estadual de Campinas, Campinas, Brazil.
authors.forename: Marcos N
authors.initials: MN
authors.lastname: Eberlin
authors.affiliation: 1 Campus Centro Oeste, Federal University of São João Del Rei, Divinópolis, Brazil; and 2 Departamento de Bioquímica and 3 Departamento de Química, Universidade Estadual de Campinas, Campinas, Brazil.
authors.forename: Paulo A
authors.initials: PA
authors.lastname: Granjeiro
authors.affiliation: 1 Campus Centro Oeste, Federal University of São João Del Rei, Divinópolis, Brazil; and 2 Departamento de Bioquímica and 3 Departamento de Química, Universidade Estadual de Campinas, Campinas, Brazil.
authors.forename: Sérgio
authors.initials: S
authors.lastname: Marangoni
mesh_terms: D000595:Amino Acid Sequence; D003553:Cystine / Q000737:chemistry; D007887:Fabaceae / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D010940:Plant Proteins / Q000737:chemistry*; D020033:Protein Isoforms / Q000737:chemistry; D012639:Seeds / Q000737:chemistry*; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014361:Trypsin Inhibitors / Q000737:chemistry*
chemical_list: D010940:Plant Proteins; D020033:Protein Isoforms; D014361:Trypsin Inhibitors; D003553:Cystine
keywords: 2D PAGE; ESI-QTOF; MALDI-TOF MS; plant Kunitz inhibitor