THE MANUFACTURE OF HEMOGLOBIN 65 



So far as I can appraise the motives of those who have concerned 

 themselves much with the manufacture of haemoglobin, I discern 

 two distinct points of view, which I may describe as the chemical 

 and the biological. In the end of course they meet, but in essence 

 the chemist wishes to obtain the material in some form in which he 

 can describe it exactly, and be sure that successive samples which 

 he may obtain are identical in composition. To this end the high 

 road is crystallisation. That reagents such as alcohol or ether should 

 be used in the manufacture of the crystals is a small matter, the danger 

 of using them is that the crystal produced may be different from the 

 haemoglobin as it exists in blood — that is relatively trifling, the im- 

 portant point is that you do get a crystal — a thing which can be 

 washed, which has a definite shape, whose angles are of definite size. 

 This, from the chemist's point of view, is something from which a 

 start can be made. If a stable standard solution of such crystals can 

 be obtained and the properties of such a standard solution described, 

 the worker may proceed to compare it with haemoglobin as found 

 in blood. 



I will describe, then, some methods which have been used for -the 

 preparation of haemoglobin. They do not include various "salting 

 out methods" which the reader can easily find in books of reference. 



Of the more modern methods the first to which reference may be 

 made is that of Christian Bohr (2). It was essentially the procedure 

 adopted by Roberts and myself (3) in 1909: 



The solution of haemoglobin was made as follows: Dog's blood, freshly drawn, was 

 whipped, centrifugalised, and the serum drawn off, Ringer's fluid was then added, to 

 make up to the original bulk, and the corpuscles shaken up. The corpuscles were given 

 three washings with Ringer's fluid in all and then ether added and the mass of cor- 

 puscles stirred until a crystalline paste was obtained; this was allowed to stand in 

 the cold over night. The etherial mass was then warmed up to 40° and centrifugalised 

 in warm tubes. The supernatant fluid and the upper portion of the crystalline layer 

 were discarded, the lower portion was slowly dissolved, with constant shaking, in 

 distilled water at 40° until the etherial smell had disappeared and only the charac- 

 teristic smell proper to dog's blood remained. The solution so prepared was filtered 

 through several layers of filter paper on a Nutsche filter and divided into two portions 

 A and B. Portion A was freed from salts as far as possible by dialysis. The essential 

 difficulty about this operation arose from the time which it took, seeing that there 

 must be no bacterial action in the solution. The following course was adopted. 

 A stoppered glass cylinder of about 10 inches in height and 3 inches in diameter 

 was employed for the dialysis. It was thoroughly rinsed with formalin and the 

 formalin removed by repeated washings with boiled distilled water. The haemoglobin 

 solution was placed in a parchment tube of the ordinary form. This tube was suspended 



BII 5 



