THE MANUFACTURE OF HEMOGLOBIN 67 



pressure of the haemoglobin itself. In the course of the dialysis the 

 corpuscles were laked and the haemoglobin became reduced. The thick 

 syrup remaining was then removed, quickly centrifuged to remove 

 the debris and then transferred to a saturator and aerated by rota- 

 tion when crystals of oxyhsemoglobin separated out. These were 

 purified by centrifuging off the mother liquor, adding distilled water 

 and placing in a vacuum, when the crystals went into solution. These 

 could be thrown out again by re-oxidation. At each resolution, 

 however, a certain amount of insoluble brown material remained 

 and had to be removed by the centrifugal machine. 



(2) Heidelberger's method. I am indebted to Dr Michael Heidel- 

 berger for the following notes : 



The method now proposed depends upon observations that suspensions of washed 

 dog or horse red cells crystallise rapidly and almost completely in the presence of 

 toluene when saturated with carbon dioxide and oxygen, and that the resulting 

 oxyhaemoglobin may be recrystallised by solution with the aid of sodium carbonate 

 and reprecipitation with carbon dioxide. 



The use of toluene was found to hasten markedly the crystallisation of the oxy- 

 haemoglobin in the corpuscles owing to its disintegrating effect on the cells them- 

 selves. While its haemolytic action is slower than that of ether, its use obviates the 

 chief disadvantages of the latter, namely, solubility in water, and the presence of 

 peroxides and other reactive substances which may alter oxyhaemoglobin. 



The carbon dioxide shifts the reaction in the acid direction past the isoelectric 

 point of oxyhaemoglobin, so that the crystals obtained are oxyhaemoglobin uncom- 

 bined with alkali. By thus promoting the crystallisation of the oxyhaemoglobin the 

 acidification also aids in the original disintegration of the corpuscles. Saturation with 

 pure carbon dioxide would drive oxygen out of the solution and change the oxyhaemo- 

 globin to the reduced form, which is too soluble to crystallise readily. In order to 

 obviate this diflficulty, one part of oxygen is mixed in a cylinder with four of carbon 

 dioxide for the saturation. Such a mixture may be passed through oxyhaemoglobin 

 solutions indefinitely without reduction. 



Removal of the salts is accomplished by the simplified form of pressure dialysis 

 suggested by Adair, Barcroft and Bock (7), ajter the desired number of recrystallisa- 

 tions has been carried out. Two recrystallisations have been deemed sufficient in this 

 laboratory, but for many purposes the oxyhaemoglobin will undoubtedly be found 

 pure enough after the first recrystallisation. On the other hand, the losses involved 

 in each recrystallisation, while appreciable, are not sufficiently large to preclude three 

 or even four recrystallisations. 



Three precautions have been found essential: 1. All operations are carried out in 

 the cold, centrifugation of the solutions being an exception if a centrifuge in a cold 

 room is not available. 2. The oxyhaemoglobin is not allowed to become dry, owing 

 to the resultant change, noted by Bohr (2), into a modification in which the oxygen 

 is not reactive. 3. During the various manipulations on the acid side of the isoelectric 

 point, before the final dialysis, care is taken to have an excess of carbon dioxide 



5-2 



