Purificação de ricina, caracterização amperométrica de atividade, e novos candidatos a inibidores

Abstract

Castor bean (Ricinus communis L.) is an oilseed of high economic value with a growing global market for castor oil at an annual rate of about 4.3%, with the expectation of a global market estimated at US $1470 million for 2025. The presence of ricin, a heterodimeric phytotoxin found in the endosperm of castor seeds, requires caution in its use, due to the extreme toxicity of this protein fraction. In parallel to the risks of handling castor in the biofuel and polymers industry, ricin is classified by international authorities as a potential agent for use in bioterrorism, since there is no pharmacological antidote for its lethal action approved by government agencies. In this work, a new assay for detecting ricin and its enzymatic activity centered on the purification of herring sperm DNA (hsDNA, herring sperm DNA) was reported by electro-oxidation of the analyte on an unmodified electrode surface. In parallel, we also sought to evaluate parameters of interaction and inhibitory potential of new candidates for toxin inhibition, respectively by spectrofluorimetry and by the defined electrochemical procedure. The results showed that the isolation of ricin from modification reported in the literature showed a satisfactory purity for the protein and high cytotoxicity. Tests based on t square wave voltammetry suggest that the release of adenine catalyzed by ricin exhibited a biphasic behavior, with a pre-stationary state preceding the Michaelis-Menten behavior, and with kinetic constants of 75 µmol.L-1 for Km and value 19 µmol.L-1 .min-1 Kcat. Studies have shown that the new method for detecting ricin is relatively simple, employs unmodified carbon paste electrodes, and can be adapted to various sources of suspect samples and detection devices. The ligands selected as possible targets for ricin showed significant values for the formation constants of the Kb complexes (22.6 ± 3.3 to 111.3 ± 7.4 x104L.mol-1 ). The thermodynamic parameters obtained with variation in pH and temperature suggest that the formation of the complexes was entropically directed, with an interaction mechanism predominantly related to the hydrophobic effect. The molecules showed better inhibition values compared to commercially obtained inhibitors and may represent a promising alternative in inhibiting the catalytic activity of ricin.