CHARLES E. LANE 173 



minimum of water and recentrifuged. The supernatant solutions 

 from these two centrifugations are combined and lyophiUzed. 



The lyophihzed "crude" toxin has regularly assayed between 

 15 and 16'i nitrogen by micro-Kjeldahl. All tests for polysaccharide 

 have been negative. A sample of crude toxin was hydrolyzed in 

 6N HCl, and the hydrolysate was analyzed on the Beckman Spinco 

 amino acid analyzer with the results shown in Table I. 



The lyophihzed toxin is lethal to mice at dosage levels of 1.7 

 mg. kilogram. 



When crude toxin was chromatographed one-dimensionally with 

 80^ »-propanol as the solvent system, a series of nine spots ap- 

 peared when the paper was developed with ninhydrin. Each of 

 the spots was separately eluted and assayed for total activity in 

 the fiddler crab, Uco piigilafor. Four of the spots accounted for 

 95% of the total biological activity of the crude toxin. 



The active regions on the chromatographic papers were eluted, 

 hydrolyzed, and rechromatographed. Each was shown to contain 

 more than one amino acid. 



Since this chromatography had been accomplished in the pre- 

 sence of a solvent and at room temperature it was felt that consid- 

 erable loss of activity may have occurred. Such an attenuation 

 might be sufficient to mask activity in other fractions. Accordingly, 

 the crude toxin was next fractionated on the Beckman refrigerated 

 paper curtain electrophoresis apparatus, using phthallate buffer pH 

 5.8 at 2". Four fractions were separated; after dialysis and lyophili- 

 zation they were carefully diluted to their relative concentration 

 in the original toxin and bioassayed on Uca pugilator. The 

 results are shown in Figure 3. 



One peptide nearly equals the activity of the original whole 

 toxin, although representing less than 10% of its weight, and it 

 therefore appears that some inert masking protein materials may 

 have been removed by electrophoresis. Physalio toxin, therefore, 

 appears to he a relatively simple protein consisting of only a few 

 toxic peptides. Our future studies will seek to describe the precise 

 molecular configuration of these peptides and to relate biochemical 

 structure to pharmacologic activity. 



I may be permitted to speculate briefly about the origin and 

 synthesis of Physalia toxin. The gastrodermis lining the battery 



