HAEMOGLOBIN 191 



and E, and the other nearly coinciding with E and b (fig. 57, spectrum 9). 

 The spectrum of alkaline haematin reappears for a short time after vigorous 

 shaking with air. 



7. Haematoporphyrin. To some strong sulphuric acid in a test-tube add a 

 few drops of undiluted blood, and observe the spectrum of acid haemato- 

 porphyrin (iron-free haematin) (fig. 57, spectrum 10). Map out all the spectra 

 you see on a chart. 



8. The photographic Spectrum. Haemoglobin and its compounds also 

 show absorption bands in the ultra-violet portion of the spectrum. This 

 portion of the spectrum is not visible to the eye, but can be rendered visible 

 by allowing the spectrum to fall on a fluorescent screen, or on a sensitive 

 photographic plate. In order to show absorption bands in this part of the 

 spectrum very dilute solutions of the pigment must be used. 



In order to demonstrate these bands, the telescope of a large spectroscope 

 is removed, and a beam of sunlight or of light from the positive pole of an 

 arc lamp is allowed to fall on the slit of the collimator. The spectrum is 

 focussed on a fluorescent screen. 1 The slit is then opened very widely, and 

 the coloured solution is interposed on the path of the beam falling on the 

 slit. 



Oxyhaemoglobin shows a band (Soret's band) between the lines G and H. 

 In haemoglobin, carbonic oxide haemoglobin, and nitric oxide haemoglobin, 

 this band is rather nearer G. Methaemoglobin and haematoporphyrin show 

 similar bands. 



The two preceding figures show the ' photographic spectra ' of haemo- 

 globin, oxyhaemoglobin, and methsemoglobin, and will serve as examples of 

 the results obtained. I am greatly indebted to Prof. Gamgee, to whom we 

 owe most of our knowledge on this subject, for permission to reproduce these 

 two specimens of his numerous photographs. 



9. Preparation of Pure Oxyhaemoglobin. The following method is described 

 in Stirling's ' Practical Physiology.' Centrifugalise dog's defibrinated blood 

 and pour off the serum. Centrifugalise again with physiological saline 

 solution repeatedly- until the supernatant fluid contains only traces of protein. 

 Mix the magma of corpuscles with two or three volumes of water saturated 

 with acid-free ether ; the solution becomes clear. Then add a few drops of 

 1 -per- cent, solution of acid sodium sulphate till the mixture looks tinted like 

 fresh blood, owing to the precipitation of the stromata. These can be 

 separated by filtration. Pour off the clear red fluid : cool it to C., add 

 one-fourth of its volume of absolute alcohol previously cooled to C. Shake 

 well, and then let the mixture stand at 5-15 C. for 24 hours. As a rule 

 the whole passes into a glittering crystalline mass. Filter at C. and wash 

 with ice-cold 25-per-cent. alcohol. Redissolvethe crystals in a small quantity 

 of water, and recrystallise as before. The crystals may then be spread on 

 plates of porous porcelain, and dried in a vacuum over sulphuric acid. 



1 Fluorescent screens, similar to those in common use in observations made 

 with Rontgen rays, may be made by coating white cardboard with barium platino- 

 cyanide. 



