320 IMMUNO-CATALYSIS 



ity of pneumococcal hemolysin appears to be controlled by the oxi- 

 dation-reduction state of certain thiol groupings in its structure. An 

 attempt has been recently made by Cohen, Halbert and Perkins 

 (1941) to obtain pneumolysin in purified form. In this work, the 

 authors carried out extensive experiments on the optimal conditions 

 of hemolysin production, method of purification, and chemical and 

 physical properties of the relatively purified product. It could be 

 precipitated at pH 4.0 without losing its activity. The purified sub- 

 stance had a nitrogen content of 1 3 to 1 5 per cent. It gave protein tests 

 and contained 1.0 to 2.0 per cent phosphorus. Commercial trypsin, 

 chymotrypsin, pepsin and papain destroyed the hemolytic activity. 

 The activity of hemolysin was unaffected by repeated extraction with 

 dry benzol, pyridine, chloroform, acetone or petroleum ether at 20° 

 to 25°. By the ultracentrifugal method, the lysin's rate of sedimentation 

 seemed to be appreciably faster than that of egg albumin (mol. wt. 

 of about 45,000) and somewhat slower than that of hemoglobin (mol. 

 wt. about 66,000). 



The hemolytic unit, H.U., was arbitrarily set at the 50 per cent 

 hemolysis of 4 ml. of a 1 per cent suspension of red cells (or the com- 

 plete hemolysis of 2 ml. of the cell suspension). The reagent red cells 

 were rabbit erythrocytes washed thrice by centrifuging in the cold with 

 M/15 phosphate of pH 7.6 and resuspended in the proportion of 1 

 volume to 99 of the same buffer. 



The activity of the purified hemolysin was measured by Cohen et al. 

 and it was found that the most active preparations contained 6000 

 H.U. per mg., or 1 H.U. per 2.4X10-^ mg. N (or 1.58X10"'' 

 mg. protein); the average sample contained 2000 H.U. per mg. 



Gelatin, casein and egg albumin exercised no inhibitory effect on 

 the lysin. The apparently clear "hemoglobin" solution, prepared by 

 laking and centrifuging washed rabbit erythrocytes, inactivated hemo- 

 lysin; but Seitz-filtered hemoglobin solution was ineffective. Benzol 

 extraction also removed the inhibitory substance from the unfiltered 

 "hemoglobin" solution. A suspension of 2 micrograms of washed 

 stromata obtained from laked rabbit blood inactivated or adsorbed 16 

 H.U. of hemolysin (2.37 microgram) in 15 minutes. The same 

 stromata exhaustively extracted with benzol after drying did not at 

 all affect, even in amounts up to 250 micrograms, the activity of 18 

 or 5 H.U. of lysin. 



