Caracterização bioquímica do inibidor de tripsina purificado da semente de tamarindo (Tamarindus indica L.) e avaliação do seu efeito na secreção de colecistocinina e leptina em modelo de obesidade experimental

Abstract

Obesity is one of the non-communicable chronic diseases with a great impact on public health. The tamarind (Tamarindus indica L.) seed has been studied for its trypsin inhibitor and one of the attributes of this partially purified inhibitor (TTI) is its relationship with satiety, increasing cholecystokinin (CCK) in eutrophic and reducing leptin in obese animals. In this study the ITT was purified, characterized and evaluated for its properties against CCK and leptin in obese Wistar rats. For the purification and characterization of this inhibitor, the crude protein extract was fractionated with ammonium sulfate followed by trypsin-Sepharose affinity chromatography, two-dimensional electrophoresis (2-DE) and High Efficiency Liquid Chromatography (HPLC). TTIp molecular mass was determined by MS-ESI. Partial sequencing of TTIp was established by MALDI-ISD. Inhibitory specificity, stability to temperature and pH was characterized for TTIp. Plasma CCK and leptin was evaluated in rats submitted to oral gavage with TTIp (730 μg / kg) comparing them to untreated obese rats. TTI was purified by HPLC with a single protein peak (ITTp). After refinement of the methods, two protein fractions were observed: Fr 1 and Fr 2, with average mass of [M+H]+ = 19594.690 Da and [M+H]+ = 19578.266 Da, respectively. The presence of 4 cysteines was estimated after reduction and alkylation of Fr 1 e Fr 2. The protein fractions showed 54 and 53 amino acid residues with exactly the same sequence. TTIp presented resistance to temperature variations, reducing about 30% of its anti-tryptic activity at 100 ºC, and resistance to pH extremes. TTIp IC50 was 2.7 x 10-10 mol.L-1 and Ki was 2.9 x 10-11 mol.L-1. The 2-DE revealed spots with isoelectric points between pH 5 and 6, and a spot near pH 8. TTIp characteristics are compatible with trypsin inhibitors from the Kunitz family. In the in vivo experiment, ITTp action on leptin reduction was confirmed, but no effect on CCK was observed in animals with obesity, corroborating previous studies using unpurified TTI. Biochemical knowledge of this molecule and the in vivo experiments shown here are novel and provide essential information for a biomolecule of possible biotechnological application.