SINGLE-STEP PURIFICATION OF CROTAPOTIN AND CROTACTINE FROM CROTALUS-DURISSUS-TERRIFICUS VENOM USING PREPARATIVE ISOELECTRIC-FOCUSING

We describe the isolation of crotoxin, a presynaptic B-neurotoxin, as well as its subunits B (crotactine) and A (crotapotin) from lyophilize d Crotalus durissus terrificus venom by a single-step preparative isoe lectric 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 st ep 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 am ino acid analysis. Crotactine, a 12-kDa protein, presented hemolytic a nd phospholipase A(2) activity. Thus, using isoelectric focusing we si multaneously purified both toxins in high yields. This method can be u sed as an alternative for the purification and characterization of pro teins from other snake venoms under conditions in which biological act ivity is retained.