SEPARATION OF 2 ISOFORMS (SER(7) THR(7)) OF NATURAL SARAFOTOXIN-A BY CAPILLARY ELECTROPHORESIS - MASS-SPECTROMETRY AND SYNTHESIS/

Each isoform was identified within the mixture in the natural sarafoto xin-a: Ser(7) and Thr(7)-SRTX-a has been identified by chemical sequen cing and recently by the sequencing of c-DNAs encoding for the sarafot oxins family. Mass spectrometry (MS) of the natural SRTX-a displayed t he presence of two isoforms as noted by chemical and c-DNA sequencing. In order to evaluate the role of the 7th residue in the bioactivity o f SRTX-a, we carried out the separation of Ser(7) and Thr(7)-SRTX-a fr om the natural SRTX-a isolated from the venom successicely by gel filt ration, ion-exchange and reversed-phase HPLC. The capillary electropho resis in micellar conditions (MECC) enabled us the separation of the S er(7) Thr(7)-SRTX-a in an analytical range of peptide only. Due to the low range of the CE process in the quantitative recovery of two isofo rms, each isoform was then synthesized by solid phase for the bioassay s and further spectroscopic studies. The MW and migration time of each synthetic isoform were found to fit with the corresponding natural pr oduct. 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 i soforms because of its speed, nanogram scale sensitivity.