Toxins, fractionation

The chemical studies of ciguatera have been particularly frustrating—shortages of research materials, purification difficulties, instability of some isolated toxin fractions, extremely small quantities present in ciguatoxic fish, lack of a reliable highly-sensitive assay to follow the isolation and purification of toxins, the complex mixtures present in toxic fractions and the complex structures of the toxins. I admire the accomplishments Professor Scheuer and others have made in spite of these obstacles. 

Prior to our understanding, there was only one reported experiment on the biosynthesis of PSP, in which C-amino acid precursors were fed to the culture of Gonyaulax catenella, and the incorporation of some radioactivity was observed in the crude toxin fraction (4). 

Our first attempt was the feeding of commercially available [guanido-l C]-L-arginine to the cultures of Gonyaulax tamarensis (Ipswich strain). The toxin fraction was isolated and further fractionated to the pure toxins (7). Figure 1 shows an example of the elution pattern of the toxins from a Bio-Rex 70 column. A good correlation between the toxicity and radioactivity was observed. 

Figure VI. Toxicity Profile from Bio-Gel P-2 Column. 1st Peak mobile phase = water 2nd peak mobile phase = 0.1 M acetic acid. 1st peak = 2 anionic, unnamed toxins. Fractions from 2nd peak pooled and re-chromatographed in Fig. V I I, (neo-STX and STX). (from Auger, 1983)...

The ultrafiltration was done in two steps, first through XMIOOA and then through PM 10 membranes at approx 50 psi in a cold room (5°C). The dialy-sate (<10,000 Dalton mol wt) was evaporated in vacuo to 300 mL and loaded onto a Bio-Gel P-2 column (5.0 x 70 cm) after adjustment of the pH to 5.5 with dilute NaOH solution. The column was first washed with 700 mL of water, and the tetrodotoxin denvatives were eluted with a 0.03-0.06 N acetic acid gradient (total 1000 mL). The combined toxin fractions were rechromatographed again in the same system. Upon completion of the second chromatography run, water-soluble components other than tetrodotoxin denvatives were mostly removed. 

The toxin fraction (FTX) of A. aperta spider venom can be also used as a P-type Ca channel blocker. In fact, this toxin was initially used to describe and characterize P-type Ca channels in Purkinje cells [4,29,30], Although FTX was initially considered to be selective for P-type Ca " channels, later it was shown to block other ionic channels [31],... 

P-type Ca channels were first described by Llinas et al. [29] in cerebellar Purkinje cells, in which Ca currents were resistant to blockade by DHPs and co-conotoxin GVIA. The toxin fraction from the venom of the funnel web spider A. aperta (FTX) was found effectively to block this resistant current, and these results led these authors to suggest the existence of a new subtype of HVA Ca " channel, which was termed P (for Purkinje ). 

Llinas R, Sugimori M, Lin JW, Cherksey B. Blocking and isolation of a calcium channel from neurons in mammals and cephalopods utilizing a toxin fraction (FTX) from funnel-web spider poison. Proc Natl Acad Sci U S A 1989 86(5) 1689-93. 

Duarte CB, Rosario LM, Sena CM, Carvalho AR A toxin fraction (FTX) from the funnel-web spider poison inhibits dihydropyridine-insensitive Ca channels coupled to catecholamine release in bovine adrenal chromaffin cells. J Neurochem 1993 60(3) 908-13. 

Subsequent examination of a large spot yielded on careful fractionation a complex with a molecular weight approximately equivalent to double that of either toxin. The NMR spectrum was roughly compatible with that of the addition of the hemiacetal of helminthosporol to what would be expected for the hemiacetal of helmintho-... 

Research in this area advanced in the 1970 s as several groups reported the isolation of potent toxins from P. brevis cell cultures (2-7). To date, the structures of at least eight active neurotoxins have been elucidated (PbTx-1 through PbTx-8) (8). Early studies of toxic fractions indicated diverse pathophysiological effects in vivo as well as in a number of nerve and muscle tissue preparations (reviewed in 9-11). The site of action of two major brevetoxins, PbTx-2 and PbTx-3, has been shown to be the voltage-sensitive sodium channel (8,12). These compounds bind to a specific receptor site on the channel complex where they cause persistent activation, increased Na flux, and subsequent depolarization of excitable cells at resting... 

Venom from the globiferous pedicellariae of sea urchins is lethal to mice, rabbits, crabs, lobsters, and worms 70). Seasonal changes in toxicity of such toxins 71) have been observed. The LD q estimate (mice) for toxic fractions from the urchin Tripneustes gratilla ranged from 0.05-0.5 mg/kg 70). 

Detoxification. The process by which bacterial toxins are converted to harmless toxoids. Formalin is used to detoxify the toxins of both Corynebacterium diphtheriae and Clostridium tetani. The detoxification may be performed either on the whole culture in the fermenter or on the purified toxin after fractionation. 

A vaccine consists of a suspension of live (attenuated) or killed (inactivated) microorganisms (in whole or fractions), whereas a toxoid is a detoxified bacterial toxin that has the ability to trigger the production of antitoxin once administered into the body. 

Imai, I., et al., Monitoring of DSP toxins in small-sized plankton fraction of seawater collected in Mutsu Bay, Japan, by ELISA method relation with toxin contamination of scallop. Mar. Pollut. Bull., 47, 1-6, 114, 2003. 

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