h. m. jope and J. R. p. o'brien 



free medium. A. A. Green, E. J. Cohn and M. Blanchard 7 were 

 the first to use crystalline human HbCO for solubility studies, and 

 R. K. Cannan and J. Redish 8 described their methods for obtaining 

 crystalline human Hb0 2 in bulk, which they developed for purely 

 practical transfusion purposes. While the work described below was 

 in progress, D. L. Drabkin 9 ' 10 , published photographs and a crystal- 

 lographic description of human Hb0 2 crystals from a study on a 

 petrographic microscope : this crystallographic description has now in 

 some respects been superseded by x-ray data on the preparations 

 described below (M. F. Perutz 11 ), and the study has been extended 

 to HbCO, MetHb and Hb, and the corresponding human foetal 

 haemoglobin derivatives. 



EXPERIMENTAL METHODS 



Red blood cells (from out-dated bank blood) were separated from the 

 plasma, washed 4 times with 1-5 per cent sodium chloride solution, 

 haemolyzed with an equal volume of water and treated with a sus- 

 pension of Cy aluminium hydroxide to facilitate the removal of stroma. 

 The aluminium hydroxide was removed by centrifuging and the 

 purified Hb0 2 solution dialyzed in the cold in sacs against either 

 65 per cent saturated (NH 4 ) 2 S0 4 or 2-8M potassium phosphate 

 pH 6-7, with frequent changes of the buffer until crystallization began 

 (about 24 hours). Crystals of adult HbCO, MetHb and Hb were 

 prepared by the same method as for adult Hb0 2 crystals. HbCO 

 was obtained by treating the red blood cells with carbon monoxide 

 and keeping the HbCO solution always in an atmosphere of carbon 

 monoxide. MetHb was prepared by treating Hb0 2 with K 3 Fc(CN) G , 

 the excess of which was removed by dialysis. Hb was prepared either 

 by treating Hb0 2 with ferrous citrate or by allowing an Hb0 2 solution 

 to become deoxygenated by bacterial action. The Hb solution and 

 crystals were always kept in an atmosphere of nitrogen. 



Foetal HbCO, Hb0 2 , MetHb and Hb crystals were prepared in the 

 same way as the corresponding adult crystals but usually only 1-2 ml 

 of blood was available and crystallization was carried out in small 

 test tubes with cellophane tied over the end in place of the cellophane 

 sac. 



RESULTS 



The crystal forms were as follows : — 



Adult HbCO orthorhombic 



Hb0 2 orthorhombic 



MetHb orthorhombic 



Hb monoclinic and another form whose 



system is still to be determined. 



270 



