182 



J. E. O'Hagan 



'essentially ionic' bonds, it could be assumed that as the haematin was being 

 removed at increasing acidity the reaction could, for practical purposes, be 

 regarded as ionic. Since the haematin would be likely to have a tendency to 

 polymerize, with drop in absorbance, it might well act as an 'indicator' of 

 the suppression of the ionization of the group ligating it. 



To rule out the effects of linkages through the haematin propionate groups, 

 aetiomyoglobin, the properties of which have been described by O'Hagan and 



0-5- 



0-3- 



01 



3 4 5 6 7 8 

 pH 



Fig. 4. Soret absorbance increment curve for ferriaetiomyoglobin at 393-5 m/n 

 prepared as described in text, o — O — O phosphate buffer, • — • — • phthalate 



buffer, I ppte., theoretical curve for acid with pK = 5-3, ■ — ■ — H curve 



for nickel mesoporphyrin + same cone, of apomyoglobin for comparison. 

 (Buffers, I = 0-05, T = 21°C.) 



.- ; i i / / / 



/ / 7 



George (1959) was utilized. It was prepared by adding 0-5 ml of 3 X 10~^ m 

 aetiohaemin in methanol to 5-5 ml of 2-9 X 10~*m apomyoglobin at pH 6-5 (no 

 added buffer) and standing at 1°C overnight. A solution prepared by adding 

 0-5 ml aetiohaemin solution to 5-5 ml distilled water was treated throughout 

 in the same manner as the aetiohaemin-apomyoglobin solution, to act as a 

 control. Next day the solutions were spun at 15,000^ for 15 min (to remove 

 free aetiohaematin) and the supernatant added in 0-2 ml ahquots to 5 ml 

 portions of buffer solution. After standing 6 hr at 1°C and 3 hr at 21°C the 

 absorbances were read (10 mm cuvette). The curve showing the absorbance 

 increment due to the apomyoglobin is shown in Fig. 4. The nature of the 

 buffer, phthalate or phosphate, made little difference to the shape of the 

 curve in the range pH 5-5-6-8. At about pH 4-95 a discontinuity in the 

 curve appeared, probably due to the taking up of the aetiohaematin by 

 carboxyl groups liberated in the protein. The curve drawn through the 

 points represents a theoretical curve for a group ionizing with pK = 4-95, 

 and for comparison curves for a group with pK = 5-3 and for the attachment 

 of nickel mesoporphyrin to apomyoglobin are given. While by no means 



