MECHANISM OF CIIOLEGLOBIN FORMATION 481 



peroxide, hydrogen donors are required for the formation of choleglobin and 

 verdohemochrome. Hydrogen peroxide alone destroys ferric hematin com- 

 pounds with complete decoloration, and a colorless ring above the green zone 

 has, indeed, been occasionally observed by McLeod and Gordon. A colorless, 

 hemolyzed ring further away from the colony and surrounding the green zone 

 is also often observed on unhemolyzed blood agar plates. 



The system in bacterial autolysates which forms green pigment 

 was studied by Hart and Anderson {111^1). They found that the 

 system is destroyed by aeration, that added hydrogen peroxide 

 destroys the green pigment, and that added catalase does not prevent 

 the formation of green pigment; they concluded from this that 

 hydrogen peroxide does not play a role. This does not necessarily 

 exclude hydrogen peroxide, however, as a factor in the formation of 

 green pigment. An excess of reducing substances, able to react with 

 hemiglobin and to reduce it back to hemoglobin, is required, in 

 addition to hydrogen peroxide in small concentrations; catalase does 

 not prevent formation of choleglobin by hydrogen peroxide formed 

 by notatin {1501). 



If the viridans effect (on unhemolyzed cells in blood agar) is due to the 

 same enzyme system, hydrogen peroxide formed by it must enter into the 

 reaction, since the enzyme does not leave the bacterial cell, nor would it 

 readily penetrate the erythrocyte membrane. It is nevertheless possible that 

 diffusible bacterial products, similar to ascorbic acid, react directly with 

 intracorpuscular oxyhemoglobin. The hydrogen peroxide formed by notatin 

 transformed intracorpuscular oxyhemoglobin only into hem/globin, not into 

 choleglobin (1501). 



The viridans effect is thus due in principle to the primary formation of a 

 hemoglobin -hydrogen peroxide compound, and to the presence of reducing 

 substances which keep the heme iron in the ferrous state. The hemoglobin - 

 hydrogen peroxide compound may be formed either by the action on hemo- 

 globin of free hydrogen peroxide — produced by a system reacting with 

 oxygen — or by direct interaction of diffusible reducing substances with 

 oxyhemoglobin. It is influenced by a variety of factors: the relative rates of 

 reaction of reducing substances with oxygen or oxyhemoglobin, the perme- 

 ability of the erythrocyte membrane, partial hemolysis (which often accom- 

 panies the viridans effect), diffusion rates of reducing substances and oxygen, 

 and the killing of the bacteria by aerobiosis. These factors decide whether 

 oxyhemoglobin is transformed into hemoglobin (as in the initial stages of 

 the action of E. rnli in bulk solutions, or by anaerobic bacteria in the deeper 

 layers), into hem/globin, or into choleglobin, or is completely destroyed with 

 the formation of colorless products. Pneumococcus, for example, reduces 

 hem/globin less rapidly in the presence of glucose, than strict anaerobes 

 {2027). 



From the investigations of Neill and Avery as well as those of Petherick 



