(g. solute/100 g. solution), and d is the density 

 (g./ml.) of the solution (table 5). 



The optical activity measurements have sev- 

 eral points of interest. Fii'st, the sample of sub- 

 stance II is optically active and is dextrorota- 

 tory. Some loss of optical activity seemed to 

 occur during the purification scheme (sample 2a 

 versus 2b, table 5). This loss may be due to 

 the removal of a volatile component, as indi- 

 cated by the further loss of optical activity ac- 

 companying evaporation (sample 2b versus 3). 

 It appeared, qualitatively, that there was no loss 

 of toxicity associated with the loss of optical 

 activity. 



CHROMATOGRAPHY 



TLC (thin-layer chromatography) and paper 

 chromatography (Heftmann, 1967) were used 

 to characterize the toxin and to determine if 

 the purified toxin contained one component or 

 two, as suggested by molecular weight versus 

 weight data from the empirical formula. 



When an Eastman chromatogram (for TLC) 

 with silica-gel absorbent was used, only one 

 spot was located (with ultraviolet light) with 

 two different solvent mixtures. The Rf values " 

 indicate that the effect of change of solvent 

 polarity was small. The behavior of the toxin 

 with TLC (silica gel) was consistent with 

 column chromatography, i.e., only one fraction 

 was obtained. 



Table 6. — Rf values for toxin chromatograms ' 



Solvent 

 Chromatogram Toxin sample mixture Rr - 



TLC (silica gel) ... Substance II (bloom) - A 0.89 



Do do B .90 



Paper Substance I (bloom) C .72 



Do do D .63 



Do Substance II ( bloom) C .83. .38 



Do do D .85, ..52 



1 Solvent mixture A, 10 percent (v/v) chloroform-absolute ethanol: 

 solvent mixture B. 10 percent (v/v) chloroform-isoamyl alcohol: C, 

 absolute ethanol: D. isoam.vi alcohol. 



-The Rf value is defined as the ratio of the distance traveled by a 

 substance (Rt) to the distance traveled by the solvent (Ri.). 



Another system, paper chromatography, was 

 selected to see if more than one component was 

 present in the fraction. Descending paper 

 chromatography was effected with diflferent 

 solvents (table 6). The chromatograms were 

 run for 5 hours, after which the paper strips 



were dried, developed with a mixture of 

 minhydrinphenol methyl cellosolve mixture, and 

 washed with dilute sodium hydroxide solutions. 

 Two components were indicated (fig. 2, table 

 6). Rf values for one component seemed inde- 

 pendent of solvent polarity ; the Rt values for a 

 second were affected by change in solvent 

 polarity. 



ACETYLCHOLINESTERASE 

 ACTIVITY 



A potentiometric determination (Jensen- 

 Holm, Lausen, Milthers, and Moller, 1959) 

 was used to measure enzyme activity. The 

 brains of five sheepshead minnows, Cyprinodon 

 variegatus, were placed in 12 ml. of distilled 

 water, cooled in an ice-water bath, and homog- 

 enized for 40 seconds with an ultrasonic 

 macerator operated at full power. The test solu- 

 tion was prepared by diluting 2.0 ml. of the 

 homogenate to 4.5 ml. with distilled water. The 

 pH was adjusted to 7.8 with 0.02 N sodium 

 hydroxide solution, and 0.05 ml. of substrate 

 (0.1 M acetylcholine iodide) was added. The 

 cholinesterase activity was determined poten- 



S 10 



TIME IMINUTESI 



15 



•> The Rt value is defined as the ratio of the distance traveled by a 

 substance (Rt) to the distance traveled by the solvent (Rl). 



Figure 3. — Potentiometric determination of cholines- 

 terase activity, micromoles of sodium hydroxide 

 added as a function of time (minutes). Curve 1, 

 standard run; curve 2, 0.02 ml. of toxin solution 

 added at point a; cui-ve 3, 0.4 ml. of toxin solution 

 added at point b; curve 4, spontaneous acid liberation 

 in absence of homogenate. 



CHEMICAL AND PHYSICAL PROPERTIES OF TWO TOXINS FROM RED-TIDE ORGANISM 



439 



