Nov. 19. igai Hemoioxins from Parasitic Worms 417 



To each of four tubes of defibrinated blood (3 drops of physiological 

 salt solution plus 2 drops of blood) a small quantity of the powder was 

 added, and the tubes were shaken thoroughly and placed in an incubator 

 at 37° C. After 2 hours hemolysis was produced in all tubes. Two lots 

 of cattle blood from different animals were collected in a 2 per cent 

 solution of sodium citrate (about 2 volumes of blood to i volume of a 2 

 per cent sodium citrate). Tested against dry Bustomum phlebotomum 

 powder the unwashed citrated blood became hemolyzed in about 2 hours 

 at 37°. 



Small quantities of powder were also tested on each of fotur lots of 

 washed cattle blood corpuscles with positive results. Hemolysis set in 

 rapidly and was complete after i hour at 37° C. 



A few drops of a 3 per cent suspension of washed sheep corpuscles 

 were hemolyzed by a small quantity of Bustomum phlebotomum powder. 

 Similar results were obtained with washed rabbit erythrocytes. 



Bustomum phlebotomum powder extracted in physiological salt solution 

 yields but a small quantity of hemolysin, as the following experiments 

 will show. 



Eighty-five mgm. of powder were suspended in 5 cc. of physiological 

 salt solution. A few drops of chloroform were added as a preservative. 

 The mixture was kept at a temperature of 35° to 37° C. for 2 days and 

 then filtered. The clear filtrate was tested on a 5 per cent suspension of 

 washed rabbit cells. Equal parts of filtrate and suspension of cells 

 yielded negative results. It was necessary to add 10 drops of filtrate to 

 3 drops of corpuscle suspension to produce hemolysis. Evidently the 

 hemolysin is firmly bound to the parasite material and is but slightly 

 soluble in salt solution. In fact, the powder which had been extracted 

 was dried and retested on rabbit blood cells, which it hemolyzed rapidly. 



An alcoholic extract of fresh specimens of Bustomum phlebotomum was 

 found to be decidedly hemolytic. The extract was prepared as follows : 

 About 100 specimens were washed a number of times in physiological 

 salt solution after they had been removed from the host. The specimens 

 were then triturated in a mortar and extracted in about 2 volumes of 

 95 per cent alcohol for about a week at 37° C. The alcohol was sepa- 

 rated from the worm material by filtration. The filtrate was evaporated 

 and the residue was shaken with a small quantity of physiological salt 

 solution, in which it dissolved, producing an opalescent solution. Tested 

 on sheep red blood corpuscles this solution produced hemolysis. A 

 quantity of the solution which hemolyzed 5 drops of a 5 per cent suspen- 

 sion of washed sheep corpuscles in about 2 hours at 37° failed to produce 

 hemolysis on an equal quantity of blood corpuscles in 20 hours in a 

 refrigerator (8°), thus showing that low temperatures paralyze the 

 action of the hemolysin. Likewise, normal horse serum (2 drops) inhib- 

 ited hemolysis of 5 drops of washed sheep corpuscles to which sufficient 

 hemolytic solution had been added to cause hemolysis in the absence of 



