Purificação, caracterização físico-química e bioatividade de uma proteína inativadora de ribossomos de Jatropha podagrica Hook

Abstract

Ribosome inactivating proteins (RIPs) are toxins with enzymatic activity rRNA- N-glycosidase. These proteins inhibit protein synthesis by specifically cleaving the adenine residue 4324 (A 4324 ) from ribosomal RNA 28S of the ribosomal

subunit 60S. This process stops the protein synthesis, leading to cell death. RIPs can be classified in type 1, 2 e 3. The main goal of this work was to isolate e characterize Jatropha podagrica Hook RIP and test its different in vitro biological activities. After collecting, cleaning the seeds, producing a flour and removing the lipids, the best protein extraction condition and purification protocols were determined. For the crude extracts (CE) the best buffer were Tris-HCl 20 mM pH 7,4. After that, a proteins precipitation with (NH 4 ) 2 SO 4 at 60% saturation was performed and denominated Fr0/60. The Fr0/60 was used for size exclusion chromatography (Sephadex G-100). After chromatography, it was obtained only one type 1 RIP, named podagrin. The podagrin showed rRNA-N-glycosidase activity and when submitted to dichroism circular analysis, podagrin presented a conformational pattern with predominance of  sheet secondary structures. It was also performed with podagrin an anti-helminth assay, against Ascaridia galli. The most efficient concentration was 5 mg/mL of podagrin, with a 100% mortality of the parasite after 72 h. With these data, it is possible to conclude that podagrin is a type 1 RIP.