SUMMARYCytotoxic material ('prymnesin') excreted into the growth medium by the phytoflagellate Pymnesium parvum was partially purified and crudely characterized. It proved to be a highly potent haemolysin; its solubility properties and some other characteristics are those of a lipid, perhaps a saponin. The high haemolytic activity which characterized the toxin was often also accompanied by a high ichthyotoxic activity. A method of locating haemolytic activity in a paper chromatogram is described. Fish were killed not by prymnesin alone, but were rendered highly susceptible to prymnesin poisoning by a range of cofactors: streptomycin sulphate, calcium chloride, magnesium chloride. Ichthyotoxicity in cofactor + prymnesin systems conformed within a wide concentration range to the relationship: a x b = Q, where a is a prymnesin concentration, b a minimum concentration of cofactor which elicits a lethal effect in presence of a, and Q is a cofactor-specific constant. The recognition of this relationship has enabled a sensitive assay to be developed. Practical implications are discussed and the hypothesis proposed that prymnesin acts on fish by inducing a state of abnormal permeability of the gill. A 2-week-old culture was used in the proportion of 1 : l O for inoculum. Largescale preparations were made from samples of pond water (batch size about 50 1.) which had been transferred during prymnesium bloom to a tank which was maintained a t ambient temperature (10-2S0) under fluorescent lighting for 1 to 2 weeks, in which time the toxic activity reached its peak.Detection and meamremend of hamolytic activity Rkfmmce 8tadurd. Digitonin (Merck, C. P.) served as a standard for reference; the stock solution was at a concentration of 0-026 % (w/v) in water. At a dilution of 1/200 in isotonic sodium chloride, this effected 50 % lysis of bovine erythrocytes within $5 min. in conditions of a standard assay described below.
STRUCTURE OF SERRATAMIC ACID 669 obtained was applied to a paper along with L-serine and authentic a-alanine and the chromatogram run in 80% phenol. Development with ninhydrin showed the product to have the same RF value as a-a]anine. 2:3-Epoxydecanoic acid. Freshly distilled octyl aldehyde (5.7 g.) and ethyl chloroacetate (5.5 g.) in dry ether (100 ml.) were added to sodium methoxide (from 10 g. of Na) suspended in dry ether. After standing for 48 hr. at room temp. water was added, and the ether layer was separated and dried over anhydrous Na2SO4. The oil left after removal of the solvent was purified by vacuum distillation, yielding ethyl 2:3-epoxydecanoate (6 g., b.p. 90-93o/0.1 mm.). Saponification rapidly occurred on the addition of an excess of 12% (w/v) KOH in methanol at room temp., with the * Part 2: Hestrin & Schramm (1954). For a preliminary communication on someexperimentsdescribedinthepresent paper and results obtained in a tracer analysis of cellulose production, see Schramm, Gromet & Hestrin (1957).
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