STUDIES ON OXIDATION AND REDUCTION BY PNEUMOCOCCUS : IV. OXIDATION OF HEMOTOXIN IN STERILE EXTRACTS OF PNEUMOCOCCUS

In the present work on oxidation and reduction by sterile extracts of pneumococcus, the preparations employed contain among other constituents, a hemolytic substance the properties of which have been described by Cole (1, 2) in his studies on pneumococcus hemotoxin. Pneumococcus extracts prepared by the methods described are actively hemolytic, 0.005 cc. of extract causing complete lysis of 2.5 cc. of a 1 per cent suspension of red cells from rabbit blood. This hemolytic property of pneumococcus extracts is destroyed by 10 minutes exposure to 55 degrees C. When pneumotoxin-containing extracts are protected from the action of molecular oxygen, their hemolytic activity remains unimpaired for considerable periods of time. In the presence of air, on the other hand, the stability of the hemolytic substance depends upon whether the particular type of extract contains a "complete" or "incomplete" oxidation-reduction system. Sterile broth extracts of unwashed pneumococci are reactive with molecular oxygen, and as a result of this union peroxide is formed whenever these extracts are exposed to air. The hemolytic activity of "complete" extracts of this type is rapidly decreased and finally destroyed in the presence of molecular oxygen. On the other hand, the "incomplete" type of extract prepared by saline extraction of washed pneumococci may be exposed to air with little or no loss of hemolytic power. This "incomplete" washed cell extract, unless reactivated, does not undergo autoxidation in the presence of air; under these circumstances peroxide is not formed and the hemolytic activity of this type of extract is not impaired by exposure to air. The stability of the hemolytic agent in the "incomplete" type of extract is evidence that this substance is itself not reactive with or affected by molecular oxygen, even in the presence of the cell enzymes. The destruction of the same hemolytic substance in extracts capable of undergoing autoxidation may be ascribed to the action of some peroxide formed by the union of molecular oxygen with easily oxidized or autoxidizable substances of the extract. It is now known that a peroxide, having the reactions of hydrogen peroxide, accumulates in sterile pneumococcus extracts during oxidation. It has been shown in the present study that the addition of preformed hydrogen peroxide destroys the hemolytic activity of pneumococcus extracts, although higher concentrations were required than were detected in oxidized extracts themselves. These facts and the known action of superoxides in analogous types of reaction make it seem not unlikely that the active agent in the destruction of pneumotoxin in oxidized cell extracts may be a peroxide; either hydrogen peroxide or some higher organic peroxide formed during autoxidation of the extract.