The concept of two-dimensional electrophoresis-guided purification proven by isolation of rhodocetin from Calloselasma rhodostoma (Malayan pit viper)

Abstract

Two-dimensional gel electrophoresis (2DE) is an important tool for investigating the complexity of snake venom proteomes. Apart from applications based on whole proteome analysis, we suggest that 2DE can be used as an assay to guide the progress of protein purification. The aim of this study was to prove the feasibility of this concept by using it to purify rhodocetin from Calloselasma rhodostoma venom. Rhodocetin (α subunit) spot on the 2DE profile of C. rhodostoma venom was first identified and confirmed by mass spectrometry, with a molecular mass of 16 kDa and calculated pI of 5.16. Rhodocetin was subsequently purified by successive anion-exchange and gel filtration chromatography. Every peak from both chromatography profiles was collected and tested on 2DE. The presence of rhodocetin (α subunit) spot in the 2DE profile of the peak DP2 indicated the presence of the protein. The purified compound was used to spike the crude venom. A spiked spot with a 1.6-fold increase in intensity was observed and its position matched to that of rhodocetin (α subunit) on the 2DE profile. Together, these spots confirmed the identity of the purified compound as rhodocetin. Hence, our results have demonstrated the effectiveness of the concept we now term 2DE-guided purification.

Comparative analysis of the venom proteome of four important Malaysian snake species

“…The 2-DE map of these venoms will play a vital role in the purification of their proteins by utilizing the 2-DE-guid purification applications as described by Tang et al [ 42 ].…”

The Role of Conventional Two-Dimensional Electrophoresis (2DE) and Its Newer Applications in the Study of Snake Venoms

“…In view of all these mentioned challenges in rhodocetin purification, we attempted to find an alternative to bypass the requirement of a bioassay to purify rhodocetin. As such, we hypothesized that a concept known as "2DE-guided purification" would be able to achieve this purpose (Tang et al, 2011). The concept of 2DE-guided purification is essentially using the presence of a protein spot on the 2DE gel as an indication of the presence of a protein within a particular sample.…”