Crotamine in Crotalus durissus: distribution according to subspecies and geographic origin, in captivity or nature

Abstract

Background: Crotalus durissus is considered one of the most important species of venomous snakes in Brazil, due to the high mortality of its snakebites. The venom of Crotalus durissus contains four main toxins: crotoxin, convulxin, gyroxin and crotamine. Venoms can vary in their crotamine content, being crotamine-negative or -positive. This heterogeneity is of great importance for producing antivenom, due to their different mechanisms of action. The possibility that antivenom produced by Butantan Institute might have a different immunorecognition capacity between crotamine-negative and crotamine-positive C. durissus venoms instigated us to investigate the differences between these two venom groups.

Methods: The presence of crotamine was analyzed by SDS-PAGE, western blotting and ELISA, whereas comparison between the two types of venoms was carried out through HPLC, mass spectrometry analysis as well as assessment of antivenom lethality and efficacy.

Results: The results showed a variation in the presence of crotamine among the subspecies and the geographic origin of snakes from nature, but not in captive snakes. Regarding differences between crotamine-positive and -negative venoms, some exclusive proteins are found in each pool and the crotamine-negative pool presented more phospholipase A₂ than crotamine-positive pool. This variation could affect the time to death, but the lethal and effective dose were not affected.

Conclusion: These differences between venom pools indicate the importance of using both, crotamine-positive and crotamine-negative venoms, to produce the antivenom.

Keywords: Antivenom; Rattlesnake; Snake venom; Toxins; Venom variation.

Figure 1.. Subspecies of Crotalus durissus analyzed in the present study: (A) C. d. terrificus, (B) C. d. collilineatus, and (C) C. d. cascavella.

Figure 2.. Percentage of C. durissus crotamine-positive (red) and crotamine-negative (blue) identified by: (A) each methodology, (B) according to subspecies and (C) to the origin of snakes. WB: western blotting; Cdt: C. d. terrificus; Cdc: C. d. collilineatus; Cdv: C. d. cascavella; CP: captivity snakes; NC: newcomer snakes. *p < 0.0003; **p < 0.0001; ***p < 0.02.

Figure 3.. Geographic distribution of C. durissus crotamine-positive (red) and crotamine-negative (blue) in a region of southeast of Brazil. ▲: C. d. terrificus; ●: C. d. collilineatus.

Figure 4.. Electrophoretic profile of crotamine-positive pool (+) and crotamine-negative pool (-) from C. durissus venom under reduced (left panel) and non-reduced (right panel) conditions. Venom pools (20 µg) were subjected to 15% SDS-PAGE and proteins were stained using Coomassie G (GE Healthcare). Arrows point to different bands between crotamine-positive and crotamine-negative venom pools. MW: molecular weight marker (Dual Color Precision Plus Protein Standards - BioRad).

Figure 5.. Elution profiles of pools of C. durissus venom by RP-HPLC monitored by 215 nm. Samples of 3 mg of lyophilized venom pools were dissolved in 0.1% trifluoroacetic acid (TFA) (solution A) and subjected to RP-HPLC on a C18 column. Elution was performed at 1.0 mL/min by applying a gradient toward 0.1% TFA and 95% acetonitrile (solution B), as described in the experimental section. Red (+): crotamine-positive venom pool; blue (-): crotamine-negative venom pool.

Figure 6.. Polyacrylamide gel electrophoresis (15%) of peaks collected by HPLC. The numbers correspond to numbered chromatographic peaks shown in Figure 7. Peaks divided into two aliquots are represented by letters “a” and “b”. MW: molecular weight marker (Dual Color Precision Plus Protein Standards - BioRad); upper panel: crotamine-positive venom pool (+); bottom panel: crotamine-negative venom pool (-).

Figure 7.. Overall composition estimated by mass spectrometry of venom pools from C. durissus according to protein families, expressed as percentages. (A) Crotamine-positive venom pool, (B) crotamine-negative venom pool. Protein family abbreviation - CRO: crotamine; PLA₂: phospholipase A₂; CTX: crotoxin; LAAO: L-amino acid oxidase; SVSP: snake venom serine protease; SVMP: snake venom metalloproteinase; PhPt: phosphoprotein; CTL: C-type lectin. Others - globin: fragment of globin HBD, BPP: bradykinin potentiating peptide, PLI: phospholipase A₂ inhibitor; VNGF: venom nerve growth factor.

Figure 8.. Lethal dose of venom pools from C. durissus. Survival of mice of different groups according to time to death after injection of (A) crotamine-positive and (B) crotamine-negative venom pools. Different colors represent different doses (µg venom/animal).

Figure 9.. Effective dose of venom pools from C. durissus. Survival of mice of different groups according to time to death after injection of (A) crotamine-positive and (B) crotamine-negative venom pools pre-incubated with crotalic antivenom. Different colors represent different doses (µL antivenom/animal).