THE MANUFACTURE OF HEMOGLOBIN 69 



revolutions per minute until complete thawing has taken place. With tubes holding 

 about 120 c.c. each this takes about 20 to 30 minutes under ordinary conditions. 

 This freezing and centrifuging are then repeated some five to six times, by which time 

 the upper layers of the liquid in the tubes will be found to be practically colourless 

 while the haemoglobin will have crystallised out at the botton. Care must of course 

 be taken not to disturb the layers of liquid during the manipulations. When sufficient 

 crystals have been collected in this way the upper liquid is sucked off, and the adhering 

 salt solution is washed from the crystalline mass by pouring on a layer of ice-cold 

 distilled water, agitating the liquid very gently and then immediately sucking off. 

 This washing may be repeated, but must be carried out with great caution as the 

 crystals are very readily soluble. Recrystallisation may be brought about by 

 dissolving the mass in about an equal volume of distilled water and repeating the 

 freezing and centrifuging as before. The crystals are best removed from the tube 

 while still frozen, and the haemoglobin may be obtained as a dry powder by keeping 

 this frozen material for a day or two over anhydrous calcium chloride in a vacuum 

 desiccator at — 15° C. The ice then sublimes directly into the drjdng agent, but even 

 at this temperature the haemoglobin suffers a certain amount of change during the 

 drying process. 



The 'preparation of hcemoglobin without crystallisation. It is urged 

 by those who wish to have haemoglobin as it exists in the body, 

 that the mere fact of crystalhsing it is unphysiological, that crystals 

 do not occur in the red blood corpuscle, and that the formation of 

 crystals involves something more far-reaching than mere concentra- 

 tion and something which may not be reversible, that, in fact, 

 haemoglobin which has been crystallised and redissolved is not the 

 game as the original material. With this point of view I at one time 

 had a good deal of sympathy, and I still think that there is much 

 truth in the general outlook — the chief criticism to which it is open is 

 that there is too much truth in it ; for, as we shall see later, it may be 

 doubted whether any haemoglobin outside the body is governed by 

 quite the same laws as that inside. If it is governed by different 

 laws it will present different properties. Even therefore if the 

 haemoglobin in a solution be the same as that in the corpuscle, it 

 cannot be assumed that it will act in precisely the same way. 



Six or seven years ago, in the endeavour to obtain haemoglobin 

 solutions, as nearly as possible like those contained in the corpuscle, 

 we naturally tried to make them of great concentration. The observa- 

 tion has often been made that haemoglobin in the corpuscle was funda- 

 mentally different from any obtainable solution because no solution 

 could be made so concentrated as the contents of the corpuscle. 

 That observation is, I think, true of the haemoglobin of some animals 



