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.

DOI: 10.7171/jbt.15-2603-002

Cite this paper

@article{Silva2015PrimarySO, title={Primary Structure of a Trypsin Inhibitor (Copaifera langsdorffii Trypsin Inhibitor-1) Obtained from C. langsdorffii Seeds.}, author={Jos{\'e} A Silva and D{\'a}via G Pompeu and Marcus B Smolka and Fabio C Gozzo and Moacyr Comar and Marcos N Eberlin and Paulo A Granjeiro and S{\'e}rgio Marangoni}, journal={Journal of biomolecular techniques : JBT}, year={2015}, volume={26 3}, pages={90-102} }