Further studies on the isolation, characterization and structure of violacein: Purple pigment of Chromobacterium violaceum

The goal of this research was to develop a method for the isolation of violacein which would produce the pigment in its natural state as much as possible, without heat, acid, base or chemical derivations which might generate artifacts; identify an instrumental technique compatible with TLC to determine and monitor the purity of the isolated pigment; and to determine the structure of the purified pigment using modern spectroscopic methods of analysis. Chemical tools, including chromatographic and spectroscopic techniques, have been used to investigate the properties of violacein and to characterize violacein isolated from the fermentation broth of Chromobacterium violaceum. The purity of the violacein isolated from the fermentation broth was monitored with FTIR difference spectroscopy. The fractionated violacein used for NMR analysis was obtained from Silica G$\sb{60}$ F$\sb{254}$-packed columns. The solvents used to obtain the most homogeneous fractions included hexane, chloroform, and methanol. Further fractionation was obtained after the column-isolated fraction was dissolved in methanol. A precipitate formed in the methanol solution which is soluble in ethyl acetate. The methanol-soluble violacein was termed violacein A and the ethyl acetate-soluble violacein was termed violacein B. While the two violaceins are chromatographically homogeneous, FTIR analysis of violacein A and violacein B confirmed that the two violaceins are spectroscopically distinguishable. Low-resolution mass spectrometry and high-resolution electron impact mass spectrometry provided evidence for a molecular weight of 438 amu for violacein A and violacein B. The low-resolution mass spectra for the two violaceins also show two different fragmentation patterns. High-resolution electron impact mass spectrometry indicates a molecular weight of 438 amu and suggested five possible formulae for violacein A. The 500 MHz proton NMR analysis of violacein A seems to support the empirical formula of C$\sb{27}$H$\sb{26}$N$\sb3$O$\sb2$ for violacein rather than the empirical formula of C$\sb{20}$H$\sb{13}$N$\sb3$O$\sb3$ proposed by earlier workers. A revised structure of violacein is presented, and based on this structure it is proposed that violacein may function as an integral part of the cell-associated endo-toxins found in Chromobacterium violaceum.