Intraspecific Differences in the Venom of Crotalus durissus cumanensis from Colombia

Abstract

Biochemical and biological differences in the venom of Crotalus durissus cumanensis from three ecoregions of Colombia were evaluated. Rattlesnakes were collected from the geographic areas of Magdalena Medio (MM), Caribe (CA) and Orinoquía (OR). All three regionally distributed venoms contain proteases, PLA₂s and the basic subunit of crotoxin. However, only crotamine was detected in the CA venom. The highest lethality, coagulant, phospholipase A₂ and hyaluronidase activities were found in the MM venom. Also, some differences, observed by western blot and immunoaffinity, were found in all three venoms when using commercial antivenoms. Furthermore, all three eco-regional venoms showed intraspecific variability, considering the differences in the abundance and intensity of their components, in addition to the activity and response to commercial antivenoms.

Keywords: Crotalus durissus cumanensis; antivenom; venom.

Figure 1

SDS-PAGE 12.5% under reducing conditions of Crotalus durissus cumanensis venom and controls. 20 μg of protein were seeded for recombinant crotoxin B [40] and native crotoxin B; for the other samples 10 μg of protein were seeded. Std.: molecular weight standard; MM: Magdalena Medio; CA: Caribe and OR: Orinoquía region.

Figure 2

RP-HPLC chromatographic elution profiles of C. d. cumanensis venom from (A) Magdalena Medio (MM), (B) Caribe (CA) and (C) Orinoquía (OR); (D) SDS-PAGE 12.5% under reducing conditions of MM and CA fractions obtained by RP-HPLC. 5 μg of total protein from each venom were seeded. Std.: molecular weight standard.

Figure 3

Protease activity zymogram in 12.5% gels for C. d. cumanensis (C.d.c.) venoms using (A) gelatin and (B) casein as substrates. Bothrops ammodytoides venom was used as a positive control (C+). In (C) the hyaluronidase activity zymogram for the three C. d. c. using 10% hyaluronic acid substrate in 10% polyacrylamide gel. 5 μg of sample were seeded. Std.: molecular weight standard.

Figure 4

Determination of the PLA₂ activity vs. cell viability in tumor lines MCF-7 y HTB-132 for the venom C. d. cumanensis from (A) Magdalena Medio (MM), (B) Caribe (CA) and (C) Orinoquía (OR). Qualitative PLA₂ assay (D) showed the formation of translucent halos around each well. A 3 µg sample was seeded in each well. Reading performed at 24 h. Bothrops ammodytoides venom was the positive control (C+); milliQ water as negative control (C⁻); fractions (F) 8–13 of the OR venom, collected from RP-HPLC.

Figure 5

Instituto Nacional de Salud (INS) antivenom recognition against C. d. cumanensis, C. d. terrificus, crotoxin subunit B and crotamine, determined by ELISA. Each point represents the average of two measurements. MM: Magdalena Medio; CA: Caribe and OR: Orinoquía.

Figure 6

Western blot assay analysis of antivenoms against the venoms of C. d cumanensis and Crotalus simus (Sim) for the antivenom from (A) Instituto Nacional de Salud (INS, Colombia), and (B) Antivipmyn-Tri^® (Laboratorio Bioclon, Mexico). MM: Magdalena Medio; CA: Caribe and OR: Orinoquía.

Figure 7

Immunorecognition of venoms by the antivenom of Instituto Nacional de Salud. Each panel shows the relative abundance obtained by RP-HPLC of what was recovered from the affinity matrix. In panel (A) non-retained (NR) and in (B) retained (R) fractions for the venom of C. d. cumanensis from Magdalena Medio (MM), Caribe (CA) and Orinoquía (OR). Arrows show the two neurotoxic components of importance. Blue arrow indicates the fraction corresponding to crotamine, which exceeds 10% recognition for the CA venom, and the black arrows correspond to the fractions related to PLA₂, where the subunit B of crotoxin also appears, whose recognition is better for the OR venom, also above 10%. Panels (C,D) show the 12.5% SDS-PAGE under reducing and non-reducing conditions of the fractions recovered from the affinity matrices of each venom. An amount of 5 μg of protein per sample were seeded. Std.: molecular weight standard.

Figure 8

Heat map indicating immunorecognition by the Instituto Nacional de Salud antivenom for C. d. cumanensis of Magdalena Medio (MM), Caribe (CA) and Orinoquía (OR) venoms.