A. ROSSI-FANELLI 



It is first necessary to determine the solubility curve of haemoglobin 

 in ammonium sulphate solution, since haemoglobin is always present 

 in skeletal muscle. These solubility curves of haemoglobin were 

 measured either by determining the concentration of pigment photo- 

 metrically, or by measuring the nitrogen content according to the pro- 

 cedure proposed by Roche and Marquet 12 . The solubility of human 

 haemoglobin in ammonium sulphate is a function of the pH and, to a 

 minor extent, of the haemoglobin concentration itself. But in con- 

 centrations approximately equal to those found in muscle extracts 

 haemoglobin is wholly precipitated in ammonium sulphate solutions 

 of 75 per cent saturation at a /?H of 6-8 to 7-0. Under these conditions 

 myoglobin remains in solution. It was, however, not easy to separate 

 the two pigments quantitatively under these conditions, and we found 

 it advisable to increase the concentration of ammonium sulphate to 

 the point where a certain amount of myoglobin is precipitated as well. 



The technique used for the crystallization of human myoglobin was 

 as follows. The muscle, taken as soon as possible after death or 

 surgical removal, was freed from fat and subcutaneous and connective 

 tissue and passed twice through a latapie (mincer). 1-2 volumes of 

 water were added and the /?H adjusted to 6-8-7-0 with N.NaOH, using 

 a glass electrode. The mixture was kept for 12 or 14 hours in the ice 

 box, being shaken intermittently. It was filtered through linen cloth, 

 the mass of muscle tissue being carefully squeezed, and the liquid 

 extract then being filtered through paper and the pH again controlled. 

 Solid ammonium sulphate was added gradually up to 78 per cent 

 saturation, complete solution being ensured after each addition. After 

 storing for 1-2 hours at 0°C the solution was filtered, and the filtrate 

 distributed in Petri dishes of about 25 cm diameter, the depth of liquid 

 not being greater than 1 or 1'5 cm. The dishes were stored at 0°C in a 

 dry room. Crystallization began after 36 to 48 hours or sometimes 

 longer. In the first instance yellow-red, strongly birefringent spheres 

 were formed (Figure 1) ; these later developed ray-like prominences and 

 finally gave rise to typical crystals (Figures 2 to 4). 



Crystallization was allowed to continue for a further 12 to 24 hours 

 at 0°C in order to increase the crystal size. The crystals were then 

 separated by centrifugation at moderate speeds, and washed repeatedly 

 with saturated ammonium sulphate solution atpH 7-0 and at low tem- 

 perature. The iron content was checked by a method described pre- 

 viously 2 and if necessary crystallization was repeated, after dissolving 

 the crystals in the minimum amount of water. 



This procedure should be repeated until a constant iron content of 

 0-34 per cent is reached. The best results are obtained if all operations 

 are carried out at 0°C in a refrigerated room. Owing to the great 



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