Toxin purification, techniques

Strains that produced each of the five serotypes known at the time were obtained and those expressing the most toxin were selected for use in further study, although most of the research involved serotype A.2 Culture conditions required to produce maximal levels of toxin were established, and techniques appropriate for purification and concentration were perfected. An important advance was crystallization of the toxin. The preferred method of toxin purification involved an initial acid precipitation from the culture supernatants, followed by redissolving the toxin in an aqueous buffer. At that point, the addition of ammonium sulfate produced toxin in a form called crystalline. To a protein chemist of today, this term means a highly purified protein that may be suitable for three-dimensional structure determination. The... 

Research on the mode of action of ciguatera-associated toxins has advanced since the early 1980s with the improvement of separation and purification techniques. Variability between samples tested and the presence of secondary toxins has yielded conflicting reports. Early studies done by Li (1965) concluded mis-... 

Analyses for the Saxitoxins. Early methods for analysis of the saxitoxins evolved from those used for toxin isolation and purification. The principal landmarks in the development of preparative separation techniques for the saxitoxins were 1) the employment of carboxylate cation exchange resins by Schantz et al. (82) 2) the use of the polyacrylamide gel Bio-Gel P2 by Buckley and by Shimizu (5,78) and 3) the development by Buckley of an effective TLC system, including a new solvent mixture and a new visualization technique (83). The solvent mixture, designated by Buckley as "E", remains the best for general resolution of the saxitoxins. The visualization method, oxidation of the saxitoxins on silica gel TLC plates to fluorescent degradation products with hydrogen peroxide and heat, is an adaptation of the Bates and Rapoport fluorescence assay for saxitoxin in solution. Curiously, while peroxide oxidation in solution provides little or no response for the N-l-hydroxy saxitoxins, peroxide spray on TLC plates is a sensitive test for all saxitoxin derivatives with the C-12 gemdiol intact. 

Chromatography. A number of HPLC and TLC methods have been developed for separation and isolation of the brevetoxins. HPLC methods use both C18 reversed-phase and normal-phase silica gel columns (8, 14, 15). Gradient or iso-cratic elutions are employed and detection usually relies upon ultraviolet (UV) absorption in the 208-215-nm range. Both brevetoxin backbone structures possess a UV absorption maximum at 208 nm, corresponding to the enal moeity (16,17). In addition, the PbTx-1 backbone has an absorption shoulder at 215 nm corresponding to the 7-lactone structure. While UV detection is generally sufficient for isolation and purification, it is not sensitive (>1 ppm) enough to detect trace levels of toxins or metabolites. Excellent separations are achieved by silica gel TLC (14, 15, 18-20). Sensitivity (>1 ppm) remains a problem, but flexibility and ease of use continue to make TLC a popular technique. 

Screening Techniques for Detecting Toxicity. Simple toxicity screening techniques are necessary to identify toxic species and to monitor the efficacy of isolation and purification procedures used to purify toxins. Atterwill and Steele 108) have recently comprehensively reviewed in vitro methods for toxicology and so much of the following is in the nature of a general overview. 

Chemical techniques for the isolation, purification and elucidation of the structure of toxins have evolved to the extent that it is frequently a routine procedure to identify the chemical nature of a newly discovered toxin once it has been purified, although difficulties arise when the toxin is a very large polypeptide, protein, or a very complex organic molecule. However, it is sometimes found that a toxin becomes progressively more labile and stabilizing contaminants are removed by the purification processes. An example of this is Cyanea toxic material which becomes increasingly labile with each purification step 111). 

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