HAEMOGLOBIN IN NUCLEAR FRACTION OF THE LIVER 635 



In Older to hacmolyse the erythiocytcs, the fraction was lioniogcnized with 

 10 vols, distilled water and allowed to stand several hours. This last step was 

 repeated at least three times more. 



After clearing the suspension of nuclei obtained in this way by adding 3% of 

 deoxycholate, the haemoglobin bands were steadily visible in the handspectio- 

 scope. The CO-band was that of haemoglobin. The pyridinc-haemoeliiomogen 

 band was located at 567 m/u. 



The haemin was extracted with a mixture of acetone and HCl (10 ml 20% 

 HCl in 1 1. of acetone). After filtration, the acetone was evaporated in vacuo. The 

 haemin was crystallized twice from cone, acetic acid, and the crystals washed 

 with 1 N HCl. The haemin was then combusted and the solution analyzed as de- 

 scribed by BoNNiCHSEN et alS''-'^^ 



In order to know in which part of the above nuclear fraction the non-extract - 

 able haemoglobin is located, we separated the fraction, prior to haemolysing the 

 red cells with distilled water, in the counter-streaming centrifuge of Lindahl^i^) 

 in five fractions containing particles of different size and different specific gravity. 

 The fractions were examined with the phase-contrast microscope and with the 

 handspectroscope. Thereafter, the specific activity of their haemin was determined. 

 The result of one of these experiments is seen in Fig. 1. 



In five of the above experiments the liver nuclei have been isolated both in 

 aqueovis medium and in organic solvents of low polarity according to the methods 

 described by Dounce etalS^^^ and Axlfbey ef oZ.^"^The unclear preparations obtained 

 in this way were treated and analysed as described above. No difference was 

 found in the properties of the non-extract able haemoglobin of the nuclear fraction 

 prepared either in aqueous or in organic medium. 



The liver ferritin was prepared as previously described by Loftfield et alS^^^ 



In experiments with 2 — 3 kgm rabbits 8 animals were investigated. Their plasma 

 was labelled with ^^Fe as described by Ehrenstein et alS^^' and reinjected. The 

 animals were killed 3 — 24 hr after injection. 



Simultaneously, in all our experiments haemin of the circulating haemoglobin 

 was analysed as well. 



In two experiments with hens 100 ml of hen blood were incubated in vitro 

 for 3 h at 37° C with 2 ml of 3.8% ammonium citrate solution containing 2 jugm 

 labelled iron of 25 juC activity. The plasma was centrifuged off and the red cells 

 washed four times with 5 vols, or more of isotonic saline. 



The erythrocyte nuclei were prepared according to Hogeboom et alS^''^ However, 

 as the red cells are not sufficiently broken down by the homogenization procedure 

 usually employed for the disintegration of other tissues, we haemolysed the cells 

 by freezing at — 20° C and thawing them three times or more, suspensed in a mix- 

 ture of 0.25 M sucrose —0.00018 M CaClg- The nuclei were then centrifuged down 

 (International Refrigerated centrifuge head No. 269) for 10 min at 2000 r.p.m. 

 Thereafter, the procedure of Hogeboom et alS^^^ was applied. Samples were taken 

 from the whole washed red cells, the stroma-free haemolysate and from the final 

 nucleus preparation. The haemin was extracted as described above. 



RESULTS AND DISCUSSION 



(a) Experiments with rats 



The amount of ^^Fe present in one jugm of iron of the nuclear haemo- 

 globin fraction of the liver varied in experiments on rats between 

 0.6x10^4 and lOxlO" % of that injected. 



