M. J. KARVONEN 

 METHOD 



Welsh sheep were used. Solubility experiments were performed 

 (a) on samples from 24 foetuses, between the foetal age of 51 and 145 

 days ; (b) on 31 samples of lamb's blood, obtained from 9 lambs 

 between birth and the 137th day of post-natal life ; (c) on samples 

 from 18 adult sheep, the oldest being 21 years old. 



The Hb solutions were prepared as 4 per cent carboxyhaemoglobin, 

 by laking washed corpuscles with distilled water. Stroma was removed 

 by filtering with kieselguhr, and by precipitating the non-Hb proteins 

 with an ammonium sulphate concentration just below the onset of 

 the precipitation of Hb. (The ionic strength of (NH 4 ) 2 S0 4 = 4-5.) 

 The clear solutions were buffered by adding an equal volume of a 

 r/2 = 4-5 (NH 4 ) 2 S0 4 solution, made to 0-4 M strength in buffer, in 

 which the proportion of primary and secondary ammonium phosphate 

 was varied according to the pH desired. The Hb was precipitated by 

 adding weighed amounts of (NH 4) 2 S0 4 . All the operations were 

 carried out at the temperature of + 1-5 ± 0-3°C, and the samples 

 were kept under a CO-atmosphere. 



It is generally recommended that the equilibration of protein 

 solutions with the protein in the solid phase be approached from the 

 supersaturated side. It turned out, however, that with the haemo- 

 globins investigated, the contrary method, approaching the equilibrium 

 from the undersaturated side, gave more satisfactory results. The 

 haemoglobins had a tendency to form supersaturated solutions, and 

 the tendency to supersaturation was the more pronounced the higher 

 the proportion of the total Hb in solution. When the equilibrium 

 between the precipitated Hb and the solution was approached from 

 the undersaturated side, the results were less anomalous and more 

 predictable. The Hb was precipitated by adding (NH 4 ) 2 S0 4 to make 

 its concentration r/2 : 9-0, and the total ionic strength correspondingly 

 9-92. The salt was allowed to dissolve slowly, during several days. 

 Then the Hb suspension was homogenized by stirring, and measured 

 aliquots were pipetted into a series of test tubes. In a ' variable 

 solvent ' series the ammonium sulphate concentration was adjusted to 

 values varying from r/2 : 4-5 to 9-0, by adding equal volumes of 

 buffered ammonium sulphate solutions of suitable strengths. In a 

 ' constant solvent ' series varying dilutions of the suspension were 

 made in a series of tubes with equal electrolyte concentration. The 

 protein concentration was always kept small — below 2 per cent — in 

 order to minimize the disturbing effect of the uneven distribution of 

 the electrolytes between the crystal water and the mother liquor. The 

 equilibrations were carried on for a fortnight. The two phases were 

 kept satisfactorily mixed by gentle stirring. After a fortnight, no 



280 



