Separation of Two Isoforms (Ser7/Thr7) of Natural Sarafotoxin-a By Capillary Electrophoresis: Mass Spectrometry and Synthesis

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Abstract
Each isoform was identified within the mixture in the natural sarafotoxin-a: Ser7 and Thr7-SRTX-a has been identified by chemical sequencing and recently by the sequencing of c-DNAs encoding for the sarafotoxins family. Mass spectrometry (MS) of the natural SRTX-a displayed the presence of two isoforms as noted by chemical and c-DNA sequencing. In order to evaluate the role of the 7 th residue in the bioactivity of SRTX-a we carried out the separation of Ser7 and Thr7-SRTX-a from the natural SRTX-a isolated from the venom successicely by gel filtration, ion-exchange and reversed-phase HPLC. The capillary electrophoresis in micellar conditions (MECC) enabled us the separation of the Ser7/Thr7-SRTX-a in an analytical range of peptide only. Due to the low range of the CE process in the quantitative recovery of two isoforms, each isoform was then synthesized by solid phase for the bioassays and further spectroscopic studies. The MW and migration time of each synthetic isoform were found to fit with the corresponding natural product. Overall, CE (off-line in this study) with MS are the method of choice for the identification of natural products such as the SRTX-a isoforms because of its speed, nanogram scale sensitivity.