Tagetitoxin purification and partial characterization

Tagetitoxin is a non-host specific toxin produced by Pseudomonas syringae pv. tagetis in host species of the Asteraceae family and in liquid culture by certain strains of the bacterium. A purification protocol involving anion exchange and partition chromatography was developed that yielded tagetitoxin purified to homogeneity. Based on dilution end-point (nanograms injected in 50 mu L that caused just detectable apical chlorosis in sunflower seedlings), the protocol resulted in a 2200-fold purification of tagetitoxin. The dilution end-point of purified tagititoxin was 10 ng/50 mu L (295 nM) and the I-50 for inhibition of Escherichia coli RNA polymerase was 0.1 mu g/mL (147 nM). Electrospray ionization mass spectrometry in 50% methanol:H2O indicated that the molecular weight of tagetitoxin is 678. Preliminary characterization of tagetitoxin structure was performed using 1D and 2D NMR spectroscopy. The results indicate that the previously proposed structure of tagetitoxin [Mitchell RE, Coddington JM, Young H. A revised structure for tagetitoxin.