Characterization and function of trypsin inhibitor purified from tamarindseed in a model of experimental obesity
Tamarindus indica L., antitryptic, Kunitz, CCK, leptin
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 its properties against the CCK and leptin hormones in obese Wistar rats. Thus, for the purification and characterization of this inhibitor, the crude protein extract was fractionated with ammonium sulfate followed by trypsin-Sepharose affinity chromatography and High Efficiency Liquid Chromatography (HPLC). TTIp molecular mass was determined by MS-ESI. Partial sequencing of TTIp was established by MALDI-ISD. We characterized TTIp by its stability to temperature and pH,inhibitory specificityand using two-dimensional electrophoresis (2-DE). 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 by the methods, two protein fractions were observed: Fr 1 and Fr 2, with protonmolar mass of [M+H]+ = 19594.6895 Da and [M+H]+ = 19577.1732 Da, respectively. The presence of 4 cysteines was estimated after reduction and alkylation to Fr 1 e Fr 2. Both protein fractions showed 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 and Ki was 2.9 x 10-11 Mol. The 2-DE revealed spots with isoelectric points between pH 5 and 6, and a spot near pH 8. Thus, it was found to be a trypsin inhibitor of 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 the unpurified TTI. Biochemical knowledge of this molecule and the in vivo experiments shown here are novel and provide essential information for a biomolecule of probable biotechnological application.