Purified snake-venom-derived α-cobratoxin (CTX), a long α-neurotoxin, was purchased from Latoxan (Portes-lès-Valence, France). The toxin was chemically modified with phenylglyoxal using a protocol described earlier [29 ]. Introduced by Kenji Takahashi more than 50 years ago as an efficient arginyl reagent, phenylglyoxal has been applied for the investigation of complex systems and as a reagent for the chemical modification of arginine residues in proteins in the past decade [30 ]. phenylglyoxal reacts with the guanido group of arginine residues under mild conditions. The derivative is sufficiently stable and decomposes very slowly [30 ].
Shortly, the toxin (0.05 mM) was incubated for 1.5 h with an excess of phenylglyoxal (Sigma, St. Louis, Missouri, US) (100 mM) in a 1-ml alkaline solution containing 50 mM sodium pyrophosphate buffer at pH 9. Then, the mToxin was purified by fast protein liquid chromatography (FPLC) using a size exclusion column (GE Sephadex 75) with an ammonium acetate buffer (pH 7). Following purification, the solution was lyophilized and redissolved in 1 ml of water. The mass of the mToxin was verified using matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) at the mass spectrometry unit of Bar-Ilan University (Dr. Rachel Persky, Department of Chemistry).
Shortly, the toxin (0.05 mM) was incubated for 1.5 h with an excess of phenylglyoxal (Sigma, St. Louis, Missouri, US) (100 mM) in a 1-ml alkaline solution containing 50 mM sodium pyrophosphate buffer at pH 9. Then, the mToxin was purified by fast protein liquid chromatography (FPLC) using a size exclusion column (GE Sephadex 75) with an ammonium acetate buffer (pH 7). Following purification, the solution was lyophilized and redissolved in 1 ml of water. The mass of the mToxin was verified using matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) at the mass spectrometry unit of Bar-Ilan University (Dr. Rachel Persky, Department of Chemistry).