Comparison of Methods for Measuring Protein Concentration in Venom Samples.

Aleksandra Bocian,Vladimir Petrílla,Justyna Buczkowicz,Monika Petrillova,Jaroslav Legáth,Jaroslav Legáth,Konrad K Hus,Vladimir Petrílla,Marcin Jaromin,Sonja Sławek,Dawid Łysiak

doi:10.3390/ani10030448

Abstract

Simple SummarySnake 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.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.

Highlights

Snake venom evolved to immobilize and capture the prey as well as for defense against other predators
Venom contains marginal amounts of nucleosides, sugars, lipids, and inorganic ions, which are less interesting from a pharmacological point of view, as it is the protein-peptide origin that is responsible for venom toxicity [3]
Venom components are of wide interest to many research groups and have been the subject of extensive research

Summary

Introduction

Snake venom evolved to immobilize and capture the prey as well as for defense against other predators. The main components of venom include proteins, with and without enzymatic activity, and peptides. Venom contains marginal amounts of nucleosides, sugars, lipids, and inorganic ions, which are less interesting from a pharmacological point of view, as it is the protein-peptide origin that is responsible for venom toxicity [3]. As about 90% of venom components are of peptide origin, all venom analyses use increasingly fast developing proteomic techniques, including electrophoresis [8,9,10,11], immunodetection techniques [12,13,14,15], different types of chromatography [16,17,18,19]

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Methods

Conclusion