Studies on Human Adult and Foetal Haemoglobins 



Foetal HbCO triclinic (or monoclinic), system still to 



be determined. 



Hb0 2 same as foetal HbCO 



MetHb same as foetal HbCO 



Hb different from foetal HbCO, Hb0 2 and 



MetHb, possibly monoclinic 



Photomicrographs of various types of crystals obtained are given 

 in Figures 1 to 8. 



The adult haemoglobin crystals are being studied by M. F. Perutz 

 and the foetal crystals by J. C. Kendrew in the Cavendish Laboratory, 

 Cambridge. 



Crystals of HbO a prepared from blood from a patient with pernicious 

 anaemia were orthorhombic. A specimen of HbCO which had been 

 lyophil dried over concentrated H 2 S0 4 for 5i hours at room tem- 

 perature, followed by \ hour at 38 °C, produced good orthorhombic 

 crystals. 



It is clear from these results that HbCO, Hb0 2 and MetHb are 

 isomorphous, both in the adult and in the foetal series, and that in 

 both cases the crystal form is different from that of the corresponding 

 Hb. 



These differences between the Hb's and the other three corresponding 

 derivatives and between adult and foetal haemoglobins are also 

 strikingly apparent in the solubility studies carried out on these 

 crystalline materials. 



In solubility studies it is necessary to ensure that measurements are 

 made only on systems which are in equilibrium, and constant values 

 for solubilities obtained by dissolving up these protein crystals are 

 frequently different from values obtained by salting out crystals at 

 the same buffer concentration. It is important therefore that all 

 comparisons are made only on results obtained in a similar manner. 

 Moreover, amorphous material is considerably more soluble than 

 crystalline and it is therefore important to ensure that the solid phase 

 consists entirely of crystalline material. 



Human HbCO, Ht>0 2 and MetHb are very soluble even in strong 

 buffers (over 20 per cent HbCO in 2M potassium phosphate, pH 6-7) 

 producing a thick solution which is difficult to filter and of a density 

 often so similar to that of the crystals that gravitational separation 

 is not possible. Filtration must be carried out at equilibrium tem- 

 perature. Filtration was carried out here through sintered glass 

 filters using a positive pressure of oxygen or nitrogen. Hb is less 

 soluble and results are obtainable by gravitational separation. 



Equilibration of the crystals with the buffer was carried out in 

 cellophane sacs with frequent changes of buffer. The crystals were 



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