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HETEROGENEITY OF HEMOGLOBIN AND METHOD OF ISOTOPIC BIOSYNTHESIS GEORGES SCHAPIRA, JEAN-CLAUDE DREYFUS AND JACQUES KRUHl The establishment of criteria of purity of proteins is a classical and a dis- appointing problem. No protein can satisfy all the different tests of purity and even the significance of the purity of a protein is now challenged. It seems that the reverse problem, the significance of the criteria for heterogeneity, must now be discussed, and eve shall propose a method of demonstrating protein heterogeneity which we have applied to hemoglobin. When a solution of a protein is studied by physicochemical methods one may observe associations with the solvent, associations with the salts and even association of the protein with itself. On the other hand, some proteins may show dissociation. In either case the solution of a homogeneous protein appears to contain more than orate component. This difficulty and its possible solution' were discussed in Professor Derrien's paper.3 When one prepares or stores a protein some "denaturation" may occur. Chemical and biochemical methods may then reveal heterogeneity which is not native but results from the manipulations or the time lapse involved. We are never able to claim the absence of some degree of alteration from the native state and it becomes difficult to claim that a heterogeneity is native. These difficulties also arise with hemoglobin. A part of the protein may be slightly modified during its fractionation the modification possibly affecting either globirl, demonstrated by partial denaturation, or the prosthetic group, as illustrated by a partial transformation into me/hemoglobin. Thus, a new compound differing f rom the native hemoglobin is artificialyv created and apparently two hemoglobins are found. D we thought that tile use of isotopes would overcome this difficulty by pro- viding results unmodified by manipulative artifacts. We have developed a method with very general scope which can be applied to the analysis and metabolic study of many proteins and other biological compounds.4 Our method is the following: a radioactive element is incorporated either in vitro or in vivo into the protein. Then, after the protein has been isolated, purified and submitted to fractionation, the isotopic composition i.e., the specific activity (S.A.) of each fraction is measured. 1. If the two fractions show different specific activities, the presence of at least two different proteins in the native state is certain. These proteins have different metabolic behavior. 2. If the two fractions obtained always display the same specific ac- ~ These studies were supported by grants from the Caisse Nationale de Securite Sociale (France) and the Institut National d'Hygiene (France). et this paper was presented by Dr. Schapira. 201

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202 PART III. ABNORMAL HEMOGLOBINS tivities, we cannot conclude that actually more than one protein is present; one may be an artifact. A conclusion concerning heterogeneity cannot be made on this evidence alone. The validity of this method is based on two assumptions: 1.) It has been generally considered that our usual chemical fractionation methods should not separate isotopes. However, Piez and Eagles in i955 observed that incorpora- tior~ of C i-; ir~to amino acids slowed their movement on chromatographic columns with possible resultant errors. Fortunately there is an extremely close relationship between the ratio of C7'i to total carbon in the labelled molecule and the degree of resolution of the labelled from the unlabelled compounds. Perhaps the best answer to this possible criticism is the parallelism of results with radioactive hemin, globin, and iron in the biological and pathological variations which shall be presented below. 2.) We must also assume that, within certain limits, no impurities are present. This Still be discussed later. METHODS Rabbit hemoglobin was prepared according to Roche, et al.6 Human hemo- globin was crystallized according to DraLkin.7 The globin was separated according to Anson and Mirsky.8 It was col- lected ore the filter and washed until the acetone became completely colorless. The globin was lyophilized and hydrolyzed by refluxing for 48 hours in 3,000 volumes of 6 :N HC1. Hemin was precipitated from the acetone filtrate by addition of water and concentrating with heat. After precipitation it was washed by centrifugation with water and alcohol. In some experiments the heme was determined as pyridine hemochromogen, in others it was crystallized (lTischer9), weighed after evaporation of the solvent on the planchets, and counted. The DNP-glycine was isolated according to Perronei after two or three passages on celite and was controlled by paper chromatography according t Biserte and Osteux,~ and A. Levy. In some experiments the DNP-glycine was oxidized by Fan Slyke's reagents and converted to barium carbonate. In another series the radioactivity was determined directly in a thin layer with the Geiger-Muller counter, following calorimetric measurement and cor- rection for thickness. Radioactive iron in the hemin solution was measured with a scintillation counter; hemin was converted to barium carbonate. In other experiments iron, after wet combustion of the hemin, was measured as -ferrous orthophenan- throline complex. Radioactive iron was measured with a Geiger-Muller counter following electrolytic plating.~3 In vitro synthesis was obtained according to Walsh, ef al.,~4 London, ei a1.,~5 and Borsook, et al.~6 Hemoglobin labelled in vivo was obtained by injection of the labelled compound and bleeding at different times, usually the fourth . r ~ ~ ~ c ay alter injection. The various hemoglobins were subjected to several fractionation procedures.

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ISOTOPIC BIOSYNTHESIS SCHAPIRA, DREYF`US .AND KFLUH 203 H2lman cord blood hemoglobin was fractionated by: 1. ~ alkali denaturation, according to Singer, et al.,l; and 2.) ion exchange chromatography, according to lIuisman arid Prins.iS We separated both an "adult" and a "foetal" fraction. Adult rabbit hemoglobin was studied by: 1.) electrophoresis, according to Tiselius' method carried out at various pH,~9 ire which we analyzed anionic and cationic fractions; 2.) progressive alkali denaturation, according to Ramsey,20 which allowed the separation of one alkali-resistant and one alkali-sensitive fraction;~3 3.) paper chromatography, which gave us, using a pH 4.3 solution as mobile phase, elongated spots of hemoglobin displaying a narrowing from which we cut and eluted the two parts of the spot ;~ and 4.) alumina chroma- tography, which enabled us to demonstrate the adsorption of about 25 per cent of rabbit hemoglobin which was easily eluted by phosphate solutions. Adolf human hemoglobin was fractionated by: 1.) alumina chromatography, (Kruh2~), by which we demonstrated the adsorption of about 10 per cent of human hemoglobin; 2.) ion exchange chromatography, according to Huisman and Prins;~8 and 3.) starch electrophoresis, according to Kunkel and Wal- lenius.23 JESUITS 1. Haman cord bloody. Partial alkali denaturation permitted the sepa- ration of foetal and adult hemoglobin containing in vitro-incorporated Fed and alpha C1I' glycine (table I). A better separation can be obtained by ion exchange chromatography. The ratios of the S.A. of the fractions were 0.25 J for the hemin and 0.47 for the globin glycine.04 ~5 TABLE I DIFFERENTIAL INTCURPCRATI^N GE FEW AND CJJ' GLYCINE INTO HEMIN ARID GLOBIN OF HE~CGT CBINS FROM HUMAN CORD BLOOD UNDER in vitro CONDITIONS Experiment Number Peso Cats Hemin Hemin (c. min/mM iron) (c. min/mM hemin/8) r/s r/s CJJI Globin ( c. min / mM glycine ) r/s 9 10 11 12 13 a 13 b 13 c 14 15 Mean 0,34 0,42 0,43 0,45 0,58 0,66 0,69 0,60 0,55 0,61 0,40 0,65 0,62 0,75 0,59 0,64 0,67 0,82 0,66 0,54 + 0,04 0,626 + 0,037 0,86 0,78 0,75 0,85 0,74 0,51 0,87 0,77 0,70 0,53 0,73 6 + 0,04

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204 PART III. ABNORMAL HEMOGLOBINS 2. Adult hemoglobin, rabbit and human. Adult rabbit and adult hu- man hemoglobins were studied after both in vivo and in vitro incorporation of radioactive iron or glycine. The hemoglobin which displayed the higher S.A. after the first stage of incorporation in vivo was called "Hemoglobin I," while that with the lower S.A. was called "Hemoglobin II." The fractions with the higher S.A. are resistant to alkali denaturation, non-adsorbed by alumina, anodic in electro- phoresis and slower moving on paper chromatography. Adult rabbit hemoglobin. More than fifty fractionations have been made of hemoglobin containing radioactive iron incorporated in vivo. A different isotopic composition in the two fractions was always observed, the ratio of specific activities always differing significantly from unity ~ table II ~ . The average ratios obtained with the various fractionation processes reported above have been: 1.11 with progressive alkali denaturation; 1.46 with electrophoresis in an alkaline buffer; 1.17 with electrophoresis in an acid buffer; 1.25 with alumina chromatography; and 1.27 with paper chromatography. The magni- tude of the ratio is an indication of the resolution obtained by the various methods. TABLE II DIFFERENTIAL INCORPORATION OF FE )0 INTO HEMCGI OBIN OF ADULT RABBITS in qJiV(J Normal Fractionation Anemic Experiment # ~ Ratio of S. A. ~ Experiment # ~ Ratio of S.A. Alkaline denaturation Paper chromatography Alumina chromatography Electrophoresis: pH 9.0-9.4 pH 5.0 10 4 11 3 1.11+0.041 6 1.27 + 0~07 1 4 1.25 + 0.06 7 1.46 + 0.19 1.17+0.05 1.22 + 0.0g 1.55 + 0.15 1.44 + 0.06 Similar results were obtained in preliminary assays using radioactive gly- cine (table IV) to be discussed later. Adult rabbit hemoglobins labelled in vitro with radioactive iron and subse- quently fractionated by alkali denaturation or by alumina adsorptions showed ratios of specific activities which are the reverse of those of fractions obtained from hemoglobin similarly labelled za vivo. Thus the in vitro kinetics of iron incorporation is the reverse of that found in viva. The experiments in which reticulocytes alone were used were exactly duplicated with a medium with blood rich in reticulocytes to which was added a suspension of bone marrow, liver extracts with hematopoletic action, vitamin Bee and folinic acid. Under these modified conditions, we found that the ratio of the specific activities of

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ISOTOPIC BIOSYNTHESISSCHAPIRA, DREYFUS AND KRUH 205 TABLE III )IFFEREN-TI.~L INCCKPCR.\TION- CF FE ~ ) IN-TO HE.~-GECEIN OF ANEMIC ADULT RABBITS 272 Intro Experiment Number Ratio of S.A. Without effectors + bone marrow + liver extract + vitamin Bee folinic acid 22 7 4 s 3 TABLE INS 0.71 + 0.31 1.33 +0.35 1.20 + 0.16 1.19 ~ 0.11 1.34 ~ 0.02 INCORPORATION OF RADIOACTIVE GLYCINE ALTO RABBIT HEMOGLOBIN to in. by - :~< A_ Ratio I / II Globin Hemin Powder Glycine . I II I 11 I 1 1 1 1 2,070 1,260 64 46 778 648 28 20.5 2,480 1,030 860 59 51 1 1 1 5,900 24,6()0 209 266 47,000 116.500 24,800 35,400 534 724 II 1,810 78,000 163,5G0 Hemin 1.64 1.20 1.20 0.65 0.70 Fractionations were carried out by alumina adsorption (unpublished data). Three days incorporation. - Globin Powder 1.40 1.36 1.16 0.78 0.74 Glycine 1.36 0.60 0.71 the two fractions is above unity again, close to that obtained by in viva iron incorporation and the reverse of that found with blood alone in vitro. Thus, these added factors modified the nature of the synthesis, hemoglobin I syn- thesis predominating (table III). Similar results severe obtained in our pre- liminary assays after labelling with radioactive glycine (table IV). Blood samples were taken for fractionation at various times after the in- . . . section of Fess. We found that the ratio of specific activities remained con- stant between the third and sixtieth day, but the reversed ratio was observed between the third and eighth hour. In fact, hemoglobin II was the first to become radioactive several hours after the iron injection, but this radioactivity subsequently decreased whereas that of hemoglobin I continued to rise until

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206 00 75 ~0 PART III. ABNORMAL HEMOGLOBINS S.A ---fib Wh I ,\ 25 '' '`` /,, \4,- i,"' . , . lo 15 20 2S FIG. 1. Time curve of incorporation of Few into hemoglobins of a normal adult rabbit in ciao. Ordinates repre- sent specific activities of the two frac- tions in arbitrary units, the number 100 being the highest value obtained in the experiment. The numerals of the abscissa represent hours. (Repro- duced from Ciba Foundation Sympos- ium on Porphyrin Biosynthesis and Metabolism, J. & A. Churchill Ltd.) the two curves crossed and finally became parallel. Thereafter the ratio of specific activities remained constant.-7 (Fig. 1~. Adult human hemoglobin. We were able to study the synthesis of adult hemoglobin, isolated in crystalline state, in several patients, including five with acute leukemia.2S O9 In vitro experiments were also carried out with blood rich in reticulocytes.2S All of these studies were done with Few. In the synthesis of hemoglobins in persons without blood disorders we found the same pattern as was found in rabbits (table V). The pattern of incorporation of radioactive iron is different for the two fractions. The S.A. curves intersected in the same manner but the time of intersection was later in the human blood. Nevertheless, we were unable to observe any decrease of S.A. of the hemoglobin II. (:Fig. 2~. Hemoglobin synthesis in blood of patients with blood disorders was similarly studied in vitro. We found a predominance of hemoglobin II synthesis in the reticulocyte-rich blood of patients with various types of anemia. In starch electrophoresis according to Kunkel and Wallenius,~3 the main spot of adult human hemoglobin labelled in vitro with Cats glycine showed isotopic hetero- geneity of the hemin and globin. (See also the paper by Dr. Kunkel).30 I00 S.A ~ - Hb II Hb 75 SO 25 FIG. 2. Time curve of incorpora- tion of Flew into hemoglobins of a normal adult man ~n ~vi~vo. As in fig. 1, ordinate represents arbitrary units, the number 100 being the highest value obtained. Note, however, that in this figure the numerals of the abscissa represent days. (Reproduced from Ciba Foundation Symposium on Porphyrin Biosynthesis and Metabolism, J. & A. Chu rebill Ltd. ) i, 4:~ . , , ~,- O ~ 4 6 ~ DAYS

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ISOTOPIC BIOSYNTHESISSCHAPIRA, DREYFUS AND KRUH 207 TABLE V DIFFERENTIAL INCORPORATION OF FESS INTO HEMOGLOBIN OF ADULT HUMAN BEINGS in Gino Ratio of S.~. II Controls 1.20 1.29 1.26 1.30 Acute Leukemia 0.83 0.77 0.50 0.44 0.43 ( in remission ) Ire vivo, the synthesis of the other hemoglobin is usually smaller in acute leukemia. In the five cases of acute leukemia iron incorporation took place according to a modality the reverse of that seen in normal subjects (table Nib. This disorder of hemoglobin formation was still present in ~ patient in com- . . . plete remission. DISCUSSION AND CONCLUSIONS 1. Human cord blood. There is general agreement that there are at least two hemoglobins, one foetal and one adult, in human cord blood. We have observed a different metabolic pattern for each of them. Iron and carbon are incorporated into hemin and glycine is incorporated into globin to a lesser degree in foetal than in adult hemoglobin. The differences are more apparent if the fractionation is performed by ion exchange chromatography according to Huisman, the ratio for hemins being 0.25 and that for the globins 0.47. Our results prove that the rate of synthesis of adult hemoglobin is higher than that of the foetal hemoglobin. Nevertheless the total amount of foetal hemoglobin synthesized during the experiments is higher, since it accounts for 75 per cent of the total. The synthesis of foetal hemoglobin is less active than that of adult hemo- globin, but this cannot explain the greater effectiveness of cord blood than adult blood in the incorporation of radioactive iron.3i 2. Adult hemoglobin. Impurities. The present method, like the meth- od of isotopic dilution, assumes the absence of impurities with different S.A. The following discussion is generalized to include incorporation of both glycir~e (incorporated into both hemin and globin) and iron in the hemo- globins of both man and the rabbit. Possible discrepancies due to non-heminic iron may be eliminated by the controls, the S.A. of hemin and of hemoglobin being the same.

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208 PART III. ABNORMAL HEMOGLOBINS Porphyrin was not a contaminant, no fluorescence being detected in either the hemin or the hemoglobin solutions. The presence of proteins other than hemoglobin could not explain the differences of S.A. in the fractions studied. Five precipitations of the same hemoglobin with large losses did not change the S.A. The ratio of S.A. of the two fractions of rabbit hemoglobin remained constant for many days and the contamination of the hemoglobin with a protein of constant S.A. seems highly improbable. In several instances the S.A. of the hemin was measured, ex- cluding possible contaminating proteins as a source of error. Catalase and foetal hemoglobin are chromoproteins which could contami~ nate adult hemoglobin and should be discussed here. Our method of prepara- tion gave hemoglobin free of catalase activity, which is originally present at a concentration only 0.3 per cent of that of hemoglobin. Theorell, et al.32 ob- served the same order of magnitude of S. A. in catalase from erythrocytes and in hemoglobin. No evidence on the occurrence of foetal hemoglobin in adult rabbit blood is available. Less than ~ per cent of foetal hemoglobin may occur in normal adult human hemoglobin.33 On Amberlite XE-64 chromatography of adult human hemoglobin labelled in vivo with Few, less than 1 per cent emerged as a small preliminary frac- tion, which may represent the foetal hemoglobin. Its S.A. was less than that of the main fraction, which could be assumed to be the adult hemoglobin. If we submitted the major fraction to alumina adsorption the two parts had the usual ratio of S.A. Significance of heterogeneity of adult hemoglobin. Biochemical as- pects. We must discuss the significance of the heterogeneity of adult hemo- globin both from a hematological and a biochemical point of view. The same results are obtained whether the hemoglobin is labelled with carbon or with iron or if the glycine is isolated from the labelled globin. We can therefore claim that adult hemoglobin of both rabbit and man is a mixture of at least two hemoglobins which are metabolically different and whose physicochemical properties allow a true separation. The present methods of fractionation do not give complete separation of the chemical individuals, only an enrichment of each f faction being obtained. The chemical differences between the fractions are unknown as is the number of components. :For instance, as observed by Prins and Huisman,34 the apparent heterogeneity of hemoglobin as shown by chromatography on alumina depends on the experimental conditions, but the effectiveness of the separation, as we have demonstrated, may be tested by the differences of S.A. of the fractions obtained. Our method of isotopic biosynthesis reveals metabolic heterogeneity of foetal and adult hemoglobin from cord blood, and the metabolic heterogeneity of normal adult hemoglobin. It is a tool which allows us to follow the progress of fractionation and to recognize resolution. It may be applied to other pro-

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ISOTOPIC BIOSYNTHESISSCHAPIRA, DREYFUS AND KRUH 209 teins and biological compounds, having been applied by others in the study of nucleic acids.35 Its primary advantage is that it eliminates the difficulties in interpretation arising from artifact heterogeneity. Hematological aspects. l hese hemoglobins have different metabolic be- haviors. Examples of different metabolism of protein according to its location are known, as, for example, the differences between hepatic and erythrocyte catalase observed by Theorell. We have observed, with Coursaget and F. Schapira,:3~; differences in the rate of incorporation of N45-labelled glycine into cardiac and gastrocnemius myosin of the rabbit. In these cases the pro- teins were perhaps the same but present in different organs. In the case of hemoglobin eve can assert that these hemoglobins are different and we can assume that these different hemoglobins are contained in different red cells. However, a distinction is to be made between red blood cells with a brief or aberrant span of life and those with a normal life span. The finding of a temporary decrease in the radioactive iron content of rabbit hemoglobin II leads to a major conclusionthe possible existence of erythrocytes levity a very short life span. This is evidenced by the very rapid rise in the specific activity of hemoglobin II, followed by a rapid fall. (Fig. 1~. The existence of erythrocytes with a short life span would account for the findings of London West Shemin and Rittenber~` and of Neuber~er.3S An. . ~ . . . a . ~ . . , O ~ When N45-labelled glycine is given, an early peak of excretion of heavy ni- trogen is found in the stercobilin isolated from the feces. The differences which remain after the disappearance of the short-lived red cells may be explained by new, as yet unproven, hypotheses. Different red cells in a pathological state may be synthesized in loci other than the bone marrow and their presence may explain the higher ratio of S.A. in the phenyl- hydrazine anemia of rabbits* and the reversed ratio in acute human leukemia. This explanation cannot apply to either the normal state or to in vitro syn- thesis, and we have to assume the heterogeneity of the population of red blood cells,40 different red cells containing the different hemoglobins which are syn- thesized at different rates. Alternatively, the heterogeneity may be intra-cellular, the same cell syn- thesizing two or more hemoglobins at different rates from different pools of precursors. We have indirect evidence for such a mechanism; in sickle hemo- ~lobin trait both Hb A and Hb S seem to be contained in the same red cell. To summarize: We considered whether the heterogeneity of a protein like hemoglobin is or is not native. A possible answer to this question can be ob- tained by the method of isotopic biosynthesis which establishes the hetero- geneity of adult hemoglobin while it avoids the difficulties of interpretation posed by the numerous possible artifacts introduced by the physical and chem- ical techniques of fractionation. -a These facts were indirectly confirmed by Benard, Dantchev, and GaJdos.39

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210 PART III. ABNORMAL HEMOGLOBINS REFERENCES 1. Colvin, J. R., Smith, D. B., and Cook, W. H.: The microheterogeneity of proteins, Chem. Rev. 34: 687, 1954. 2. Roche, J., and Derrien, Y.: Les hemoglobines humaines et les modifications physi- ologiques et pathologiques de leurs caracteres, Le Sang 24: 97, 1953. 3. Derrien, Y.: These Proceedings. 4. Schapira, G., Dreyfus, J. C., and Kruh, J.: Metabolisme different de deux hemo- globines chez un meme animal adulte etudie a ['aide du fer radioactif, Compt. rend. Acad. sci. 230: 1618, 1950. 5. Piez, K. A., and Eagle, H.: Systematic edect of Cti-labeling on ion-exchange. Chromatography of amino acids, Science 122: 968, 1955. 6. Roche, J., Derrien, Y., and Moutte, M.: Solubilite dans les solutions salines con- centrees et caracteres specifiques des hemoglobines sanguines, Bull. Soc. chim. biol. 23: 1114, 1941. 7. Drabkin, D. L.: A simplified technique for a large scale crystallization of human hemoglobin. Isomorphous transformation of hemoglobin and myoglobin in the crystalline state, Arch. Biochem. 21: 224, 1949. 8. Anson, M. L., and Mirsky, A. E.: Protein coagulation and its reversal, J. Gen. Physiol. 13: 469, 1930. 9. Fischer, fI.: Hemin, Org. Synth. 21: 53, 1941. 10. Perrone, J. C.: Separation of amino-acids as dinitrophenyl derivatives, Nature 161: 513, 1951. 11. Biserte, G., and Osteux, R.: La chromato',raphie de partage sur papier des dini- trophenyl-amino-acides, Bull. Soc. chim. biol. 33: 50, 1951. 12. Levy, A. L.: A paper chromatographic method for the quantitative estimation of amino acids, Nature 174: 126, 1954. 13. Schapira, G., Dreyfus, J. C., and Kruh, J.: Recherches sur la biochimie de l'hemo- globine a ['aide du fer radioactif. I-Fractionnement des hemoglobines de lapin adulte par denaturation alcaline, Rull. Soc. chim. biol. 33: 812, 1951. Walsh, R. J., Thomas, E. D., Ghow, S. K., F~luharty, R. G., and Finch, C. A.: Iron metabolism. Heme synthesis in ~vitro by immature erythrocytes, Science 110: 396, 1949. 15. London, I. M., Shemin, D., and Rittenberg, D.: Synthesis of heme i~z vitro by the immature non-nucleated mammalian erythrocytes, J. Biol. Chem. 183: 749, 1950. 16. Borsook, H., Deasy, C. L., Haagen-Smit, A. J., Keighley, G., and Lowy, P. H.: Incorporation in q~itro of labeled amino acids into proteins of rabbit reticu- locytes, J. Biol. Chem. 196: 669, 1952. 17. Singer, K., Chernoff, A. I., and Singer, L.: Studies on abnormal hemoglobins. I Their demonstration in sickle cell anemia and other hematologic disorders by means of alkali denaturation, Blood 6: 413, 1951. 18. Huisman, T. H. J., and Prins, H. K.: Chromatographic estimation of four dif- ferent hemoglobins, J. Lab. and Clin. Med. 46: 252, 1955. 19. Schapira, G., Kruh, J., Bussard, A., and Dreyfus, J. C.: Recherches sur la bio- chimie de l'hemoglobine a ['aide du fer radioactif. II-Fractionnement des hemo- globines de lapin adulte par electrophorese, Bull. Soc. chim. biol. 33: 822, 1951. 20. Ramsey, M.: A comparative study of hemoglobin denaturation, J. Cell. & Comp. Physiol. 18: 369, 1941. Kruh, J., Dreyfus, J. C., and Schapira, G.: Recherches sur la biochimie de l'hemo- globine a ['aide de fer radioactif. III-Fractionnement des hemoglobines de lapin adulte par chromatographie sur papier, Bull. Soc. chim. biol 31: 773, 1952.

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ISOTOPIC BIOSYNTHESIS SCHAPIRA, DREYFUS AND KRUH 211 22. Krnh, l.: Recherches sur la biochimie de l'hemoglobine a ['aide de fer radioactif. IVF`ractionnement des hemoglobines de lapin adulte par chromatographie sur alumine. Comparaison avec les resultats obtenus par d'autres methodes, Bull. Soc. chim. biol. 34: 778, 1952. 23. Kunkel, H. G., and Wallenius, G.: New hemoglobin in normal adult blood, Sci- ence 122: 288, 1955. 24. Dreyfus, J. C., Schapira, G., and Harari, M.: Incorporation du fer radioactif in ~itro dans les p;lobules roue:es du nouveau-ne (hemop:lobine foetale et hemo- globine adulte), Compt. rend. Soc. biol. 148: 1798, 1954. 25. Schapira, G., Dreyfus, J. C., Kruh, J., Paoletti, C., and Boiron, M.: Heterogeneite metabolique de l'hemoglobine de sang de cordon, etudiee a ['aide du fer et du glycocolle radioactifs, Compt. rend. Soc. biol. 149: 1178, 1955. 26. Kruh, J., Dreyfus, J. C., and Schapira, G.: Recherches sur la biochimie de l'hemo- globine a ['aide de fer radioactif. NTBiosynthese des hemoglobines z~z ~vitro Bull. Soc. chim. biol. ]5: 1181, 1953. 27. Dreyfus, J. C., Schapira, G., and Kruh, J.: Phases initiales de l'hemoglobinopoiese etudiees a ['aide du fer radioactif, Compt. rend. Soc. biol. 147: 782, 1953. 28. Schapira, G., Dreyfus, T.- C., and Kroh, I.: Synthese des hemoglobines humaines marquees ~n ~z~o tnommes normaux et atteints de leucemie aigue) et i~z ~ztro, Compt. rend. Soc. biol. 147: 780, 1953. 29. Schapira, G., Tubiana, M., Dreyfus, J. C., Kruh, l., Boiron, M., and Bernard, ~.: Recherches sur l'anemie des leucoses aigues. IMetabolisme du fer dans la leucose aigue etudie a ['aide du Fer 59, Revue Hematol. 9: 3, 1954. 30. Kunkel, H. G.: These Proceedings. 31. Jensen, W. N., Ashenbrucker, H., Cartwright, G. S., and Wintrobe, M. M.: The uptake in qJitro of radioactive iron by avian erythrocytes, J. Lab. Clin. Med. 42: 833, 1953. 32. Theorell, H., Beznak, M, Bonnichsen, R., Paul, K. G., and Akeson, A.: On the distribution of injected radioactive iron in guinea pigs and its rate of appear- ance in some hemoproteins and ferritin, Acta Chem. Scand. 5: 445, 1951. 33. Chernoff, A. I.: The human hemoglobins in health and disease, New Engl. J. Med. 253: 322, 1955. 34. Prins, H. K., and Huisman, T. H. T.: Some observations about the heterogeneity of hemoglobin in aluminum oxide chromatography, Biochim. et bioph. acta 20: 570, 1956. 3 5. Bendich, A., Russell, P. J., Jr., and Brovvn, G. B.: On the heterogeneity of the desoxyribonucleic acids, J. Biol. Chem. 201: 305, 1953. 36. Schapira, G., Coursaget, J., Dreyfus, J. C., and Schapira, F.: Incorporation dans la myosine du glycocolle marque a l' azote. Roles de l' atrophie et de la topo- graphie musculaire, Bull. Soc. chim. biol. 35: 1309, 1953. 37. London, I. M., West, R., Shemin, D., and Rittenberg, D.: On origin of bile pig- ments in normal man, ;. Biol. Chem. 184: 351, 1950. 38. Neuberger, A.: Studies on mammalian red cells, Ciba Foundation Conference on Isotopes in Biochemistry, p. 68. j. & A. Churchill, Ltd., London, 1955. 39. Benard, H., Dantchev, D., and Gajdos, A.: Augmentation de la proportion d'hemoglobine alcalino-resistante au cours de la reparation de certaines enemies experimentales chez le lapin, Le Sang 25: 78, 1954. 40. Ponder, E.: L'idee d'heterogeneite appliquee aux globules rouges, a leurs stromas et aux cellules en general, Revue Hematol. 11: 123, 1956. . . .