Poster Presentation 11th Australian Peptide Conference 2015

ToPI1: a novel class of protease inhibitor characterized from Tityus obscurus scorpion venom (#150)

Caroline BF Mourão 1 , Guilherme D Brand 2 , Maura V Prates 3 , Carlos Bloch Jr 3 , Sônia Freitas 4 , Elisabeth F Schwartz 1
  1. Laboratório de Toxinologia, Universidade de Brasília, Brasília, Brazil
  2. Departamento de Química Orgânica, UnB, Brasília, Brazil
  3. Laboratório de Espectrometria de Massa – EMBRAPA Recursos Genéticos e Biotecnologia , Brasília, Brazil
  4. Laboratório de Biofísica Molecular, UnB, Brasília, Brazil

The interest in the identification of new protease inhibitors (PIs) has recently increased due to their potential use in the prevention of human diseases such as cancer, inflammation and bleeding. Many PIs have been characterized from venomous animals, such as sea anemones, snakes and, more recently, scorpions. In this study we present the chemical and biological characterization of a serine protease inhibitor isolated from the venom of Tityus obscurus (Buthidae), a scorpion responsible for many envenomation cases in the Amazon rainforest. Named ToPI1, it was purified by RP-HPLC from the crude venom and its partial sequence was determined by MALDI in-source decay. The complete sequence was then characterized based on the precursor obtained by the cDNA library of T. obscurus venom gland. With half-size of the common Kunitz-type PIs characterized from venomous animals, but also with three disulfide bridges, ToPI1 presents less than 45% sequence similarity with other peptides from public database. ToPI1 was synthesized by Fmoc/t-butyl solid phase strategy and oxidized in the presence of glutathione. Its interaction with surface immobilized trypsin was analyzed by Surface Plasmon Resonance. In this experimental setup, ToPI1 showed picomolar affinity to trypsin (KD of 530 pM at 25°C). The temperature presents few influence on the dissociation constant, which varies from 658 to 472 pM from 15-35°C, respectively. The ToPI1:trypsin interaction was shown to be spontaneous (ΔG = -12,60 kcal.mol-1), endothermic (ΔH = 2,28±0,84 kcal.mol-1) and entropy-driven (ΔS = 49,92±2,82 cal.mol-1.K-1). The linear peptide had insignificant affinity to trypsin, showing that the tridimensional structure is essential to activity. Analysis by circular dichroism spectroscopy (190-260 nm) suggests predominance of β-type and disordered structures. In the pH range of 3-9, ToPI1 did not denatured even at 95°C. New experiments are to be performed in order to characterize the binding surface of the ToPI1:trypin complex.

Acknowledgement: CNPq, FAPDF, Embrapa-CENARGEN.