Purification, crystallization and preliminary X-ray diffraction analysis of crotamine, a myotoxic polypeptide from the Brazilian snake Crotalus durissus terrificus

Abstract

Crotamine, a highly basic myotoxic polypeptide (molecular mass 4881 Da) isolated from the venom of the Brazilian rattlesnake Crotalus durissus terrificus, causes skeletal muscle contraction and spasms, affects the functioning of voltage-sensitive sodium channels by inducing sodium influx and possesses antitumour activity, suggesting potential pharmaceutical applications. Crotamine was purified from C. durissus terrificus venom; the crystals diffracted to 1.9 Å resolution and belonged to the orthorhombic space group I2(1)2(1)2(1) or I222, with unit-cell parameters a = 67.75, b = 74.4, c = 81.01 Å. The self-rotation function indicated that the asymmetric unit contained three molecules. However, structure determination by molecular replacement using NMR-determined coordinates was unsuccessful and a search for potential derivatives has been initiated.

Figure 1

(a) Purification of crotamine by cation-exchange chromatography (Mono S HR 10/10). Inset, Coomassie-stained 12% SDS–PAGE gel of crotamine. Lane M, molecular-mass markers (labelled in kDa); lanes 2–9, pooled purified crotamine at different concentrations. (b) Representative MALDI–TOF spectrum obtained from crotamine.

Figure 2

Native crystals of crotamine with approximate dimensions of 0.5 × 0.25 × 0.1 mm.

Figure 3

X-ray diffraction pattern of crotamine: concentric rings indicate resolution ranges and the high-resolution diffraction pattern is magnified.