ANTI-ENZYME IMMUNITY 325 



Stable at pH 4.0 to 8.5. The minimal solubility, as in pneumococcal 

 hemolysin (Cohen, et ah, 1942) in a concentrated broth, was at pH 

 4.0 to 4.5. 



The unit of hemolytic activity was taken as the smallest amount of 

 the solution tested that completely hemolyzed 0.5 ml. of a 2.5 per cent 

 suspension of sheep red blood cells in 30 minutes at 37° in a final 

 volume of 1.0 ml. The purified hemolysin contained 0.001 mg. of 

 nitrogen per hemolytic unit. The hemolysin produced in a synthetic 

 medium on purification contained 0.0005 mg. of nitrogen per hemo- 

 lytic unit. The dialyzed hemolysin contained 15.3 per cent nitrogen, 

 1.9 per cent sulfur and approximately 0.1 per cent phosphorus. Tests 

 for inorganic sulfate were negative. Tests for organic sulfate showed 

 1.0 per cent. The inactive preparations of hemolysin gave negative 

 nitroprusside tests, but when they were treated with potassium cy- 

 anide, the test became positive. Using cysteine as a standard a colori- 

 metric determination showed that only a part of the total sulfur was 

 present as -SH. 



The hemolysin was reversibly inactivated by oxidizing agents such 

 as oxygen, hydrogen peroxide, iodine and potassium ferricyanide. 

 Such oxidized hemolysin could be reactivated by sodium hydrosulfite 

 or by cysteine, glutathione, thioglycollic acid, sodium bisulfite, hy- 

 drogen sulfite or potassium cyanide. Sodium thiosulfate also had a 

 slight activating effect. The effect of potassium cyanide, in comparison 

 to cysteine, was definitely less. Only a very slight activation of hemoly- 

 sin with ascorbic acid or with ascorbic acid plus iodide was observed. 

 Hydrogen and palladium-asbestos had no activating effect. The leuco 

 forms of several dyestuffs were also unable to activate the hemolysin. 

 Rosindulin G.G., which has a very negative potential, in no case caused 

 the activation of inactive hemolysin. Equally negative results were 

 obtained with methylene blue, indigo disulfonate, indigo tetrasulfonate 

 and pyocyanine. Activation with methyl viologen, although far from 

 complete, was very definite. 



The hemolysin was also inactivated by cuprous oxide, phenylmer- 

 curic chloride, phenylmercuric nitrate, alloxan, maleic acid, iodoace- 

 tic acid, iodoacetamide and cholesterol. The inactivation caused by 

 each of these, except the last, was at least in large part reversed by 

 thiol compounds. 



The results with the activating and inactivating agents suggested 



