DANSYL-POLYMYXIN SYNTHESIS

1. DANSYL-POLYMYXIN SYNTHESIS REFERENCE: Schindler and Tueber. Antimicrob. Agents Chemother. 8:95-104, 1975. Kris-itd.unair.ac.id (for education purpose only)

2. METHOD: Make column using Sephadex G-25 in a 50 x 2.5 column. (Bed vol. approx - 100 ml, uses 10-15g dry bead.) Equilibrate column with at least 100 ml of 10mM Na2HPO4 pH7.1/0.145M NaCl buffer. Dissolve 40mgs of Polymyxin B sulfate in 1.2 ml of 0.1M NaHCO3. Dissolve 10mgs of Dansyl-chloride in 0.8 ml of acetone. Add the polymyxin to the Dansyl-chloride and place in the dark for 90 min at room temperature. After incubation load the mixture onto a Sephadex G-50 column (50 x 2.5 cm) equilibrated with 10mM Na-phosphate buffer (pH 7.1) containing 0.145M NaCl. Collect 5-6ml fractions.

3. The Dansyl-Polymyxin comes out in a fairly broad peak ahead of the unreactedDansyl-chloride peak. Location of the Dansyl-Polymyxin in the collected fractions can be determined by holding a UV lamp over the fractions and looking for fluorescence. The fluorescence of the Dansyl-Polymyxin is yellowish, while the unreactedDansyl-chloride is more blue-green. The fractions containing the Dansyl-polymyxin are extracted into about 1/2 volume of n-butanol. The butanol is then evaporated to dryness in a glass petri dish place inside a dessicator which is then evacuated and placed at temperature of 37C. This takes about 24 hrs. The dried Dansyl-Polymyxin is dissolved in 3 ml of buffer (5mM Hepes, pH 7.0) and stored in aliquots at -20C. The concentration of the Dansyl-polymyxin is determined by dinitrophenylation assay. NOTE:  DPX is now commercially available from Molecular Probes (P13238) in addition to other fluorescently labelledpolymxin B compounds.