Single-step purification of crotapotin and crotactine from Crotalus durissus terrificus venom using preparative isoelectric focusing

Abstract

We describe the isolation of crotoxin, a presynaptic B-neurotoxin, as well as its subunits B (crotactine) and A (crotapotin) from lyophilized Crotalus durissus terrificus venom by a single-step preparative isoelectric focusing procedure. From 98 mg of dried venom protein 20.1 mg of crotactine and 13.1 mg of crotapotin were recovered in the first step of focalization and 4.2 mg in a second run. These values correspond to 35.7% of the total venom protein applied. Crotactine separated in the 9.3-7.0 pH range (tubes 1-6) and crotapotin in the 1.8-2.8 pH range (tubes 15-19) and both were homogeneous by SDS-PAGE and N-terminal amino acid analysis. Crotactine, a 12-kDa protein, presented hemolytic and phospholipase A 2 activity. Thus, using isoelectric focusing we simultaneously purified both toxins in high yields. This method can be used as an alternative for the purification and characterization of proteins from other snake venoms under conditions in which biological activity is retained.

Fraction of C. d. collilineatus venom containing crotapotin protects PC12 cells against MPP + toxicity by activating the NGF-signaling pathway

“…Its venom consists of different classes of proteins and peptides like bradykinin-potentiating peptides, convulxin, crotamine, crotoxin, and gyroxin [ 7 - 10 ], crotoxin being the major component [ 11 - 13 ]. Crotoxin is a molecule composed of two subunits that are non-covalently bonded, i.e., subunit A or crotapotin (crotoxin acid chain) and subunit B or phospholipase A 2 (PLA 2 ) [ 11 , 14 - 17 ]. Crotapotin is a non-toxic, non-enzymatic protein [ 6 , 14 ] with anti-inflammatory [ 18 - 20 ], and antimicrobial activities [ 16 , 18 , 19 , 21 , 22 ].…”

Estudos estrutura-função de neurotoxinas isoladas de veneno crotalico e botropico

“…No entanto, esta metodologia tem alguns aspectos negativos que merecem ser avaliados, como a perda de material durante as inúmeras etapas e o gasto de tempo experimental. Aguiar et al (1997) propuseram um novo método, mais rápido, de purificação da crotoxina por cromatografia de isofocalização. Contudo, os resultados foram confusos, associados à quantidade reduzida de material obtido.…”