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140 PART II. BIOSYNTHESIS OF HEMOGLOBIN thesis of heme and free erythrocyte protoporphyrin, Scand. J. Lab. CIin. Invest. 7: 80, 1955. 12. Goldberg, A., Ashenbrucker, H., Cartwright, G. E., and Wintrobe, M. W.: Studies on the biosynthesis of heme in vitro by avian erythrocytes, Blood 11: 821, 1956. 13. Laforet, M. T., and Thomas, E. D.: The effect of cobalt on heme synthesis by bone marrow in ~vitro, J. Biol. Chem. 218: 595, 1956. 14. Morell, H., and London, I. M.: Unpublished data. 15. Shemin, D.: Personal communication. 16. Gabrio, B. W., Donohue, D. M., and Finch, C. A.: Erythrocyte preservation. V. Relation between chemical changes and viability of stored blood treated with adenosine, J. Clin. Invest. 34: 1509, 1955. 1/. Gabrio, B. W., Donohue, D. M., Huennekens, F. M., and Finch, C. A.: Erythrocyte preservation. VII. Acid-citrate-dextrose-inosine (ACDI) as a preservative for blood during storage at 4 C., J. Clin. Invest. 35: 657, 1956. 18. Jaffe, E. R., Lowy, B. A., Vanderhoff, G. A., Aisen, P., and London, I. M.: Proc. VI Int. Cong. of Hematology, Boston, 1956; Abstract in Federation Proc. 15: 304, 1956. 19. Prankerd, T. A. J., Altman, K. I., and Young, L. E.: Abnormalities of carbohy- drate metabolism of red cells in hereditary spherocytosis, I. Clin. Invest. 34: 1268, 1955. 20. Rubinstein, D., Kashket, S., and Denstedt, O. F`.: Studies on the preservation of blood. IV. The influence of adenosine on the glycolytic activity of the ery- throcvte during storage at 4 C.. Canad. T. of Biochem. PhYsiol. 34: 61. 1956. 21. Christensen, H. N., Riggs, T. R., and Ray, N. E.: Concentrative uptake of amino acids by erythrocytes in vitro, J. Biol. Chem. 194: 41, 1952. 22. Stern, J. R., Eggleston, L. V., Heins, R., and Krebs, H. A.: Accumulation of glu- tamic acid in isolated brain tissue, Biochem. J. 44: 410, 1949. 23. Christensen, H. N., and Streicher, J. A.: Concentration of amino acids by the excised diaphragm suspended in artificial media. I. Maintenance and inhibition of the concentrating activity, Arch. Biochem. 23: 96, 1949. 24. Riggs, T. R., Christensen, PI. N., and Palatine, I. M.: Concentrating activity of reticulocytes for glycine, J. Biol. Chem. 194: 53, 1952. 25. Thorell, B.: Studies on the formation of cellular substances during blood cell pro- duction, Acta Med. Scand. 200: (Suppl.) 1-120, 1947, (accompanies vol. 129). 26. Hammarsten, E., Thorell, B., Aqvist, S., Eliasson, N., and Akerman, L.: Studies on the hemoglobin formation during regenerative erythropoiesis, Exp. Cell Res. 5: 404, 1953. 27. Muir, H. M., Neuberger, A., and Perrone, J. C.: Further isotopic studies on haem- oglobin formation in the rat and rabbit, Biochem. J. 52: 87, 1952. DISCUSSION Dr. Felix Hazlrowitz: I just want to comment briefly on what Dr. London said about Christensen's experiments on the uptake of glycine into red blood cells. Dr. Lietze in my laboratory has repeated and confirmed these experi- ments, using radioactive glycine. About 80 to 90 percent of the intracellular glycine is free; only 10 to 20 percent is bound to protein. Dr. Kurt Salomon: I would like to comment briefly on the findings of Dr. London concerning the fact that the biosynthesis of hemoglobin can be affected in different ways. In collaboration with [ones E. Richmond and Kurt I.

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DISCUSSION 141 Aitman several years ago we investigated the ability of bone marrow prepara- tions of irradiated rabbits to incorporate the alpha carbon atom of glycine into hemin and globin.i The animals were irradiated with 800 r, and then killed at various time intervals after radiation, e.g., immediately after radia- tion, or to be more precise as soon as possible the preparation of the bone rr~arrow taking about half an hour 24 hours later, 48 hours later, etc. The bone marrow was removed from the long bones, homogenized and incubated ill the presence of alpha carbon labeled glycine. We then isolated hemoglobin and prepared from it hemin and globin. It was found that hemin synthesis decreased approximately 24 hours after radiation, and remained low for about three to four weeks. After this time period hemin synthesis increased. Globin synthesis on the other hand was not influenced markedly during the first week after irradiation, and remained on that level over the time period studied. In other words, one may say that in intact rabbits radiation affects hemin and globin synthesis differently, the biosynthesis of hemin being more radia- tion-sensitive than the biosynthesis of globin, but we do not know why. We concluded, as Dr. London does, that the biosyntheses of hemin and of globin are dissociated processes, a finding which has been confirmed by Neuberger and associates using somewhat different methods of approach. Dr. H. Borsook: I regret that in the rush of trying to get through my paper I did not refer to the much earlier work of Dr. Salomon and his associates. We knew that the two processes of heme and of globin synthesis could be separated. In the demonstration that the two processes are separable, one may overlook what to me, at any rate, is a remarkable fact that, at least in rabbit reticulocytes, the two go so closely together. There is evidently some kind of interaction. This interaction can be suspended or interfered faith. It becomes an interesting question: is this a mere coincidence in rabbit reticulocytes? I think not, because one can vary the rate of hemoglobin syn- thesis greatly and at the same time retain equal rates of synthesis of heme and oT globin. It becomes an interesting general question regarding the synthesis `~f conjugated proteins in general, namely: is there any relation between the synthesis of the protein part and of the conjugate? Dr. Salomo7~: May I add one more comment? My previous remark should be somewhat qualified for an adequate assessment of the radiation effects mentioned. It should be kept in mind that bone marrow after irradiation with the doses used is changed histologically and biochemically.3 Keeping this in mind we can only say that the ratio of the biosynthesis of hemin to globin is different from what it was before radiation of the animal, but we know noth- ing about the causes of this phenomenon. It might be that by using a better- 1.Richmond, J. E., Altman, K. I., and Salomon, K.: The effect of x-radiation on the biosynthesis of hemoglobin, J. Biol. Chem. 190: 817, 1951. 2. Muir, H. M., Neuberger, A., and Perrone, J. C.: Further isotopic studies on hemoglobin formation in the rat and rabbit, Biochem. J. 52: 87, 1952. 3. Lajtha, L. G.: Bone marrow cell metabolism, Physiol. Rev. 37: 50, 1957.

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142 PART II. BIOSYNTHESIS OF HEMOGLOBIN defined in vitro system and carrying out simultaneous cytological studies an alternate interpretation may be necessary. Dr. Jean-Cla?lde Savoie: I should like to report on some studies concerned with the site of formation of heme within the duck erythrocyte. This work was done in collaboration with Mrs. Helena ~I:orell and Dr. Irving M. London. Duck erythrocytes were incubated for varying periods of time with glycine-2-C7~. After incubation, the erythrocytes were fractionated and hemin was isolated from each fraction and its specific activity determined. The re- sults indicate that heme is synthesized in close association with particulate matter of the cell and that, with time, the newly synthesized heme passes pro- gressively into the soluble portion of the erythrocyte. Samples of duck erythrocytes were suspended in 50 ml. of isotonic NaCl. I;~errous iron as FeCl~, was added in a concentration of 1 ~ 1O-4 Molar to enhance heme synthesis. The samples in duplicate were incubated in room air faith shaking in a water bath at 37 C. To each sample glycine-~-C~J' was added as the isotopic substrate. Immediately after incubation the erythrocytes were separated from the suspension medium by centrifugation and were washed four times with three volumes of cold, isotonic saline. The packed cells were lysed with distilled water and al ter hemolvsis; 10.6 gnu. of sucrose dissolved in 10 ml. of plater svas added to reconstitute the added water to isotonicity. The hemolysate was then centrifuged for 20 minutes at 4 C. and at 23,000 ~ G and the supernatant hemoglobin solution, fraction I, was separated from the residue by Recantation. In order to remove front the residue the hemoglobin which was not firmly bound to it, the residue was washed twice. The first washing divas performed with 100 ml. of distilled H,O and the suspension was then centrifuged at 23,000 x G for 20 minutes and the supernatant solution decanted. The residue was next extracted with isotonic sodium chloride and the extract re- moved by high speed centrifugation. The water and saline washes~were com- bined (fraction II) and the washed residue constituted fraction III. lIemin was isolated from each fraction by HCl-acetone extraction and crystallized. The hemin in crystalline form was plated at infinite thickness and counted in a thin end-~vindow gas flow counter. Table I presents the results of an experiment which divas performed on a ing periods of time. The data indicate that after one half hour of incubation, the specific activity in the hemin derived from the stroma is much higher than that derived from the hemoglobin solution, the ratio of the specific ac- iarge sample of duck erythrocytes which were pooled and incubated for vary- tivities in these two fractions being close to 6. With more prolonged incu- bation, there is a progressive decline in this ratio which indicates the pro- gressive increase in the specific activity of the hemoglobin in the supernatant solution. D ~ , 1 ~

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DISCUSSION 143 Table II shows the results of separate experiments, each performed with ~ different pool of duck erythrocytes incubated with glycine 2-C for periods of time varying from one half to four hours. The results indicate that despite the different samples of blood and the performance of these experiments at different times, the ratio of the specific activity of the hemin of fraction I to specific activity of the hemin of fraction III at any given time is similar, and that there is ~ progressive decline in this ratio with time. Heme is formed in close association with the particulate matter of the erythrocyte. With longer periods of incubation, progressively larger amounts of the newly-formed and highly-labeled theme are released into the soluble portion of the cell. More precise fractionation of the cells will be performed in order to localize the sites of heme formation more definitely. TABLE I CHANGES IN SPECIFIC ACTIVITIES OF 'FLESIDUE BOUND HEMIN' AND OF IIEMIN FROM "[IEMOGLOBIN SOLUTION" WITH INCREASING INCUBATION TIMES FRACTION I FRACTION II FRACTION III Fr III RATI O Fr I 1/2 Fir. 1 Hr. A B A B 73 72 212 187 115 124 244 240 432 428 753 680 5.9 5.9 3.6 3.6 773 4 Hr. A B 792 822 860 1,510 1,630 2.0 2.0 (Counts are expressed as counts per minute per ma. of hemin). Fraction I - Hemin flom Hemoglobin Solution. Fraction II - Hemin from Combined Washes of Residue. Fraction III- Hemin from Water-insoluble Cell :Residue. A and B are duplicate samples. TABLE II RATIOS OF SPECIFIC ACTIVITIES OF RESIDUE-BOUND HEMIN (FRACTION III) TO SPECIFIC ACTIVITIES OF HEMIN OF HEMOGLOBIN SOLUTION (FRACTION I) Exp. 1 Exp. 2 Exp. 3 Exp. 4 ]/2 Hr. 1 Hr. S.8 5.5 3.2 3.4 2.9 2.8 5.9 5.9 3.6 3.6 2 Hr. 2.2 2.2 4 Hr. 1.5 1.5 2.0 2.0