An in-depth snake venom proteopeptidome characterization: Benchmarking Bothrops jararaca

Nicolau, Carolina Alves; Carvalho, Paulo C.; Junqueira-de-Azevedo, Inácio L.M.; Teixeira-Ferreira, André; Junqueira, Magno; Perales, Jonás; Neves-Ferreira, Ana Gisele C.; Valente, Richard H.

doi:10.1016/j.jprot.2016.06.029

Cited by 61 publications

(45 citation statements)

References 119 publications

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“…In the 2-D Quant Kit method, copper ions attach to the peptide bonds in the protein, so this method is independent of the amino acid composition of the proteins in the sample. This kit has been repeatedly used to measure the concentration of proteins in venom [28,39] and this is also the most commonly used method in our laboratory [10,34,35,40]. However, obtained results by 2-D Quant Kit and Bradford for A. contortrix venom show low variability, whereas for N. ashei venom, the differences are large.…”

Section: Discussionmentioning

confidence: 99%

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Comparison of Methods for Measuring Protein Concentration in Venom Samples

Bocian

Sławek

Jaromin

et al. 2020

Animals

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Simple Summary: Snake venom is mostly composed of proteins and peptides, which are of interest to many researchers due to their potential pharmacological properties. Due to their biochemical character, these components are analyzed using proteomic techniques such as electrophoresis, chromatography and mass spectrometry. A very important stage of such studies is the measurement of protein concentration in the sample, which is most often performed by colorimetric methods. In the presented article, we used five such techniques on venoms of two snake species, namely Agkistrodon contortrix and Naja ashei. In the case of A. contortrix venom, four methods provide similar concentration values, whereas, in the case of N. ashei, the differences between results are very significant. The source of these differences should probably be seen in the differences in amino acid composition of proteins of these two venoms. With this report, we would like to draw attention to the need to select an appropriate method for measuring the concentration of protein in the venom, especially in the case of Elapid species.Abstract: Snake venom is an extremely interesting natural mixture of proteins and peptides, characterized by both high diversity and high pharmacological potential. Much attention has been paid to the study of venom composition of different species and also detailed analysis of the properties of individual components. Since proteins and peptides are the active ingredients in venom, rapidly developing proteomic techniques are used to analyze them. During such analyses, one of the routine operations is to measure the protein concentration in the sample. The aim of this study was to compare five methods used to measure protein content in venoms of two snake species: the Viperids representative, Agkistrodon contortrix, and the Elapids representative, Naja ashei. The study showed that for A. contortrix venom, the concentration of venom protein measured by four methods is very similar and only the NanoDrop method clearly stands out from the rest. However, in the case of N. ashei venom, each technique yields significantly different results. We hope that this report will help to draw attention to the problem of measuring protein concentration, especially in such a complex mixture as animal venoms.

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Section: Discussionmentioning

confidence: 99%

“…In the first one, the venom is freeze-dried or dried and suspended in a specific amount of buffer to obtain samples at a specific concentration [24][25][26][27]. The second approach uses crude venom and this approach requires a step to measure the protein concentration of the sample [28][29][30].…”

Section: Introductionmentioning

confidence: 99%

Comparison of Methods for Measuring Protein Concentration in Venom Samples

Bocian

Sławek

Jaromin

et al. 2020

Animals

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“…Venomics studies of Bothrops have identified two of the most abundant protein groups: metalloproteases and phospholipases A 2 (PLA 2 s). These protein groups are responsible for the most severe local clinical manifestations that are produced by these venoms, such as hemorrhage and muscular and endothelial damage [ 14 , 19 – 22 ].…”

Section: Introductionmentioning

confidence: 99%

Heterologous expression of the antimyotoxic protein DM64 in Pichia pastoris

et al. 2017

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Snakebite envenomation is a neglected condition that constitutes a public health problem in tropical and subtropical countries, including Brazil. Interestingly, some animals are resistant to snake envenomation due to the presence of inhibitory glycoproteins in their serum that target toxic venom components. DM64 is an acidic glycoprotein isolated from Didelphis aurita (opossum) serum that has been characterized as an inhibitor of the myotoxicity induced by bothropic toxins bearing phospholipase A2 (PLA2) structures. This antitoxic protein can serve as an excellent starting template for the design of novel therapeutics against snakebite envenomation, particularly venom-induced local tissue damage. Therefore, the aim of this work was to produce a recombinant DM64 (rDM64) in the methylotrophic yeast Pichia pastoris and to compare its biological properties with those of native DM64. Yeast fermentation in the presence of Pefabloc, a serine protease inhibitor, stimulated cell growth (~1.5-fold), increased the rDM64 production yield approximately 10-fold and significantly reduced the susceptibility of rDM64 to proteolytic degradation. P. pastoris fermentation products were identified by mass spectrometry and Western blotting. The heterologous protein was efficiently purified from the culture medium by affinity chromatography (with immobilized PLA2 myotoxin) and/or an ion exchange column. Although both native and recombinant DM64 exhibit different glycosylation patterns, they show very similar electrophoretic mobilities after PNGase F treatment. rDM64 formed a noncovalent complex with myotoxin II (Lys49-PLA2) from Bothrops asper and displayed biological activity that was similar to that of native DM64, inhibiting the cytotoxicity of myotoxin II by 92% at a 1:1 molar ratio.

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“…Cerca de 90% do peso seco dos venenos é constituído por proteínas, porém, apesar de apresentarem mais de 250 proteínas distintas (NICOLAU et al, 2017), essas pertencem apenas a algumas famílias proteicas, incluindo enzimas (serino proteases, metaloproteases, L-aminoácido oxidases e fosfolipases A 2 ) e proteínas sem atividade enzimática (desintegrinas, lectinas, peptídeos natriuréticos, miotoxinas, proteínas secretadas ricas em cisteína, fatores de crescimento neural e endotelial vascular, cistatinas e inibidores de proteases do tipo Kunitz) (CALVETE;SANZ, 2007;FRY. 2005;CALVETE, 2004;MARKLAND, 1998;SERRANO et al, 2005).…”

Section: 51unclassified

Estudo e caracterização bioquímica e biológica do veneno de serpentes jovens e adultas da espécie >i<Bothrops moojeni>/i<

Hatakeyama¹

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Aos meus amigos de toda a vida, os mais presentes e os não tão presentes por motivos de trabalho ou simplesmente por morar longe. Obrigada, Gustavo, Hiago, Giovane, Marcelo e Ana Gabriela. Em especial ao Gustavo por ser quem é, pelo companheirismo que temos mesmo não sendo tão presente fisicamente, por como nossa amizade evoluiu desde que nos conhecemos, há mais de 10 anos, e também por ser quem me apresentou o Hiago, outra figura ímpar na minha vida, com quem compartilhei momentos especiais mesmo com seu gênio difícil de lidar e com um ego tão inflado quanto o maior balão que já voou no céu. São as amizades que ganhei fora do meio acadêmico e são duas das que mais considero. Aos dois, agradeço do fundo de coração todo o carinho. Amo vocês! À Dra. Kathleen Fernandes Grego, Diretora do Laboratório de Herpetologia do Instituto Butantan, por permitir a realização deste trabalho e por estar sempre disposta a nos ajudar em todos os aspectos. Ao Dr. Sávio Stefanini Sant'Anna, do Laboratório de Herpetologia do Instituto Butantan, pela extração de venenos das serpentes. A todos do laboratório de Herpetologia pelo apoio, companheirismo, simpatia e por proporcionar a este lugar um ambiente tão agradável para se trabalhar e se conviver diariamente.

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An in-depth snake venom proteopeptidome characterization: Benchmarking Bothrops jararaca

Cited by 61 publications

References 119 publications

Comparison of Methods for Measuring Protein Concentration in Venom Samples

Comparison of Methods for Measuring Protein Concentration in Venom Samples

Heterologous expression of the antimyotoxic protein DM64 in Pichia pastoris

Estudo e caracterização bioquímica e biológica do veneno de serpentes jovens e adultas da espécie >i<Bothrops moojeni>/i<

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