Identification of crotamine in the venom of Crotalus durissus collilineatus by three different methods

Oliveira, Sayonara Ay Moré de; Magalhães, Marta Regina; Salazar, Vania Cristina Rodríguez; Valadares, Marize Campos; Cunha, Luíz Carlos da

doi:10.1016/j.toxicon.2014.12.015

Cited by 10 publications

(6 citation statements)

References 33 publications

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“…We couldn't visualize the band corresponding to girotoxin which should be around 30 KDa; besides, crotamine was not displayed which should be between 5 and 10 KDa under reducing conditions. This result is in agreement with some studies conducted with C. durissus terrificus where it was discovered that not all individuals of this species have crotamine; it is known as "positive crotamine" and "negative crotamine" according to the presence of this protein (De Oliveira et al, 2015) and (Oguiura, 2009).…”

Section: Crotalus Durissus Resultssupporting

confidence: 93%

Analysis of the protein profile of the venoms of snakes Bothrops asper, Bothrocophias myersi and Crotalus durissus from the Colombian Andean Region obtained by RP-HPLC

Loboguerrero

Sarmiento

Galvis³

et al. 2021

Rev. colomb. biotecnol.

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Snake venoms comprise a highly complex mixture of proteins, and there is also a high interspecific and intraspecific variability in their composition, even in the same region. Our aim was to compare the composition of the venoms of Bothrocophias myersi, Crotalus durissus and Bothrops asper, snakes from the Andean region in Colombia by Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC). The venoms were given to the research group under an agreement with the Fundación Zoológica de Cali. The venoms pool was obtained by manual extraction, lyophilized and refrigerated. The protein found in the venoms was quantified by spectrophotometry using the Bradford and Lowry methods and direct measurement by Nanodrop®. The protein composition was stablished by RP-HPLC, using a Lichosper 100 RP, C18 column (250X4 mm) with a pore size of 5µm, as well as by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The highest quantity of protein was found in the venom of B. myersi (108,6 mg/mL) followed by C. durissus (78,1 mg/mL) and B. asper (74.1 mg/mL). All venoms showed bands of 15 and 50 KDa by SDS-PAGE; The most important finding is the abundance of PLA2 and svMP in the venom of B. myersi. Chromatographic analyses revealed a very similar venom composition profile, but also certain differences in toxins abundance. We conclude that the process of separating the venom proteins by RP-HPLC and SDS-PAGE are very important as a first step to know the venoms profiles, which in turn could allow medical staff to elucidate the clinical syndrome produced by snakebites.

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Section: Crotalus Durissus Resultssupporting

confidence: 93%

Analysis of the protein profile of the venoms of snakes Bothrops asper, Bothrocophias myersi and Crotalus durissus from the Colombian Andean Region obtained by RP-HPLC

Loboguerrero

Sarmiento

Galvis³

et al. 2021

Rev. colomb. biotecnol.

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“…Although methods such as HPLC, SDS-PAGE and ELISA have been proposed to identify and quantify crotamine [32, 33], the ANVISA guidelines neither require standardization of the crotamine concentration in venom pools, nor do they propose any analytical method for the identification and quantification of this toxin. The current ANVISA regulation recommending the use of crotamine-positive venoms for immunization, without knowledge of the actual concentration of this compound in the venom, is problematic and can lead to fluctuations in antibody concentrations among batches.…”

Section: Discussionmentioning

confidence: 99%

Antivenomics as a tool to improve the neutralizing capacity of the crotalic antivenom: a study with crotamine

Teixeira-Araújo

Castanheira

Brazil-Más

et al. 2017

J Venom Anim Toxins Incl Trop Dis

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BackgroundSnakebite treatment requires administration of an appropriate antivenom that should contain antibodies capable of neutralizing the venom. To achieve this goal, antivenom production must start from a suitable immunization protocol and proper venom mixtures. In Brazil, antivenom against South American rattlesnake (Crotalus durissus terrificus) bites is produced by public institutions based on the guidelines defined by the regulatory agency of the Brazilian Ministry of Health, ANVISA. However, each institution uses its own mixture of rattlesnake venom antigens. Previous works have shown that crotamine, a toxin found in Crolatus durissus venom, shows marked individual and populational variation. In addition, serum produced from crotamine-negative venoms fails to recognize this molecule.MethodsIn this work, we used an antivenomics approach to assess the cross-reactivity of crotalic antivenom manufactured by IVB towards crotamine-negative venom and a mixture of crotamine-negative/crotamine-positive venoms.ResultsWe show that the venom mixture containing 20% crotamine and 57% crotoxin produced a strong immunogenic response in horses. Antivenom raised against this venom mixture reacted with most venom components including crotamine and crotoxin, in contrast to the antivenom raised against crotamine-negative venom.ConclusionsThese results indicate that venomic databases and antivenomics analysis provide a useful approach for choosing the better venom mixture for antibody production and for the subsequent screening of antivenom cross-reactivity with relevant snake venom components.

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“…Our data showed that there is no significant difference among the results obtained by each methodology (Figure 2A). However, both western blotting and ELISA assay require the production of specific antibodies in laboratory animals, since they are not commercially available [50]. Thereby, a C. durissus snake selection system based on the detection of crotamine in its venom by SDS-PAGE could be implemented in the Laboratory of Herpetology at Butantan Institute to include in the serpentarium a determined percentage of crotamine-positive snakes, assuring the presence of this protein in the mixture used in antivenom production.…”

Section: Discussion Identifying Crotamine-positive and Crotamine-negamentioning

confidence: 99%

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

Tasima

Serino-Silva

Hatakeyama

et al. 2020

J. Venom. Anim. Toxins incl. Trop. Dis

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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 A 2 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.

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Identification of crotamine in the venom of Crotalus durissus collilineatus by three different methods

Cited by 10 publications

References 33 publications

Analysis of the protein profile of the venoms of snakes Bothrops asper, Bothrocophias myersi and Crotalus durissus from the Colombian Andean Region obtained by RP-HPLC

Analysis of the protein profile of the venoms of snakes Bothrops asper, Bothrocophias myersi and Crotalus durissus from the Colombian Andean Region obtained by RP-HPLC

Antivenomics as a tool to improve the neutralizing capacity of the crotalic antivenom: a study with crotamine

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

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