Comparison of Methods for Measuring Protein Concentration in Venom Samples

Abstract

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.

Different Research Approaches in Unraveling the Venom Proteome of Naja ashei

“…In this context, it seems that the venomics protocol for quantitative analysis, although laborious, remains the most accurate method available. Although the methods proposed therein are not without the limitations of quantitative methods, especially in the context of biases related to the presence of certain amino acids in protein sequences [19], the three-leveled quantification workflow still seems the best available solution [1].…”

Proteomics and life-history variability of Endogenous Phospholipases A2 Inhibitors (PLIs) in Bothrops jararaca plasma

“…Direct concentration measurement was performed on the NanoDrop™ One (Thermo Fisher Scientific) at 280 nm, with an estimated percent of extinction coefficient (ε1%) of 10. The analysis was performed in triplicate and the data was expressed in mg/mL [ 47 ].…”

Protective Mechanism of Velvet Antler Polypeptide on Myocardial Ischemia-Reperfusion Injury in Rats

“…The lysate was added to ice homogenate, the supernatant was obtained after centrifugation, and put at -80°C for later use. The protein concentration of myocardial tissue in each group was calculated by bicinchoninic acid (BCA) assay (Bocian et al, 2020). The solution volume was calculated according to the sample concentration, and the protein was denatured by boiling after adding the sample buffer.…”