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THE INCORPORATION OF GLYCINE INTO GLOBIN AND THE SYNTHESIS OF HEME IN DUCK ERYTHROCYTES AND RABBIT RETICULOCYTES~ IRVING M. LONDON,-: HELENA MORELL ID ANTON KASSENAAR-~ The studies which I should like to report were designed to investigate the formation of globin by duck erythrocytes in vitro and to compare the rates of synthesis of heme and of the incorporation or glycine into globin under different experimental conditions. The biosynthesis of heme has been extensively studied ire intact human, rabbit and avian erythrocytes,~ '' and in non-intact prepara- tions of these cells.3~5 Although evidence for the formation of peptide bonds in vitro in the hemoglobin of duck erythrocytes was obtained several years ago with the use of N75-labeled histidine,0 relatively little attention has been given to the usefulness of this system for the study of the formation of protein. Chicken erythrocytes and reticulocytes have been employed for the study of the rates of formation of heme and of hemoglobin.0 The incorporation in vitro or various isotopically-labeled amino acids into tile total protein of reticulocytes obtained from phenylLydrazine-treated rabbits leas been studied; and more recently these observations have been extended to the incorporation of glycine into globin and its utilization for the synthesis of heme in these cells.S As a system for the study of protein synthesis, or for the incorporation of an amino acid into a protein, the immature mammalian or avian erythrocyte affords the advantages of simplicity and ready availability. More important, however, is the fact that as the protein under study, hemoglobin can be iso- lated in relatively pure form and with highly reproducible analytic values. Materials and Procedures. In these experiments, the standard reaction mixture consisted of 4 ml of washed duel: erythrocytes suspended in 8 ml of isotonic sodium phosphate buffer, pH 7.4; an amino acid mixture such as described by Borsook and his associates,7 but without glycine; glucose in a concentration of 200 mg per 100 ml; penicillin G and streptomycin sulfate, 10 ma. of each per 100 ml of incubation mixture; and glycine-2-C2'' in a concentration of 1.2 mg of glycine (10 microcuries) per 12 ml of incubation mixture. In the standard incubation procedure, 50 ml Erlenmeyer flasks, con- taining 12 ml of reaction mixture, were incubated with shaking in a water bath at 37 C. for 4 hours. The reaction mixture was exposed to air and all experiments were done in duplicate or triplicate. After incubation, the samples were transferred to a cold room (4 C.) and This work was supported by grants from the Office of Naval Research (contract Nonr-1765 (00) ), the American Cancer Society and the Atomic Energy Commission. -i-This paper was presented by Dr. London. +~ Fellow of the Rockefeller Foundation 1956. Present address: Department of Endo- crinology, University of Leiden, Leiden, Holland. 131

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132 PART II. BIOSYNTIIESIS OF HEMOGLOBIN were washed three times with 10 volumes of isotonic sodium chloride. The erythrocytes were lysed with 10 ml of distilled water and the lysate was mixed thoroughly by shaking with 2 ml of toluene until a firm emulsion was formed. After centrifugation the clear layer of hemoglobin solution was removed and filtered through paper. The hemoglobin solution was then added dropwise to 10 to 12 volumes of acetone, containing 1.2 ~ of con- centrated HC1, as in the Anson and Mirsky procedure.0 The precipitated "globin" was washed several times with acid-acetone until the supernatant solution after centrifugation was colorless. The globin was then redissolved in water and reprecipitated with acid-acetone. The precipitated material was dissolved in distilled water, precipitated with 14 To trichloroacetic acid, washed twice with 7 ~ trichloroacetic acid and once with distilled water. The globin was then dissolved in 2 ml of 1N NaOH and the precipitation and washing procedures were repeated. The final preparation was dried by washing with an ethyl ether-acetone mixture (1 :1) twice with acetone and, finally, twice with ethyl ether. The final preparation is a pure white powder. Care must be exercised to keep it dry for it takes up water readily and its radioactivity may become erroneously low. This procedure has yielded uniformly reproducible results. The precise nature of the protein, which is called "globin," is not definitively established, but from experiments which are reported below, it would seem reasonable to conclude that the protein which we isolate by this method is, indeed, the "globin" of hemoglobin. Hemin was isolated from the solution of acid- acetone and was recrystallized prior to counting its radioactivity. The radioactivity of the hemin and of the globin was determined in a thin end-window gas flow counter. The results are expressed as the specific ac- tivity, counts per minute per millimole of glycine in heme or in globin. The method of calculation and the assumptions on which the calculation is based are presented elsewhere.~ The "heme to globin ratio" represents the specific activity of the glycine in heme relative to the specific activity of the glycine in globin, i.e., counts per minute per millimole of glycine in heme relative to counts per minute per millimole of glycine in globin. The data represent the means of replicate samples with a maximal range of variation of + 5~. E~idence for Parity of the Protein Preparations. Protein samples iso- lated by the method described above were shown to be of constant specific activity when the material was redissolved in 1N sodium hydroxide and the purification procedures were repeated. Furthermore, protein samples iso- lated from lysed erythrocytes which had been incubated with labeled glycine at 37 C. or from intact erythrocytes which were incubated at 4 C. con- tained no measurable radioactivity. ~lable 1 presents a compar~son ot the rad~oact~v~ty ot prote~n prepared by this method with that of globin isolated from crystalline hemoglobin in ali- quot samples. No significant differences in radioactivity were found. It ~ r

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GLYCINE INTO GLOBIN- LONDON, MORELL AND KASSENAAR 133 TA:E3LE I COMPARISON OF RADIOACTIVITY OF PROTEIN ISCLATED FROM "HEMOGLOBIN SOLUTION" AND OF GLOBIN ISOLATED FROM CRYSTALLINE HEMCGLOBIN Experiment Protein Isolated from Globin Isolated from Number "Hemoglobin Solution" Crystalline Hemoglobin c.p.m./mM c.p.m./mM 1 24,950 2S,750 2 22,300 23,250 seems reasonable, therefore, to refer to the protein isolated from the "hemo- globin solution" as globin. Incubation Medium. In preliminary experiments incubation in plasm resulted in a higher rate of incorporation of glycine into globin than incuba- t~on in 0.9C/o sodium chloride solution, isotonic sodium phosphate buffer pH 7.4 or Krebs-Ringer phosphate buffer plI 7.4. However, in order to elimi- nate unknown variable factors which might be present in plasma, isotonic phosphate buffer was chosen as the medium. There was little difference in the rate of incorporation of glycine into globin in a medium of isotonic phos- phate buffer, Krebs-Ringer phosphate buffer or 0.9 /7o sodium chloride. 7:onicity of Medium. The system for the incorporation of glycine into globin has a sensitivity to changes in the tonicity of the incubation medium as indicated in table II. On lowering the tonicity to 50% of isotonicity, the incorporation of glycine into globin is diminished by as much as 22 to 63~. Only slight hemolysis was observed at this level of tonicity. On raising the tonicity to 125570 of isotonicity, there was some diminution in the incorpora- tion of glycine into globin. TABLE II INFLUENCE OF TONICITY OF THE INCUBATION MEDIUM ON THE INCORPORATION OF GLYCINE INTO GLOBIN AND HEME Radioactivity of Glycine per cent per cent of control In Heme of control Tonicity of Medium Expt. No. per cent of Control In Globin c.p.m /mM c.p.m. /mM 1 Control 30,300 125 25,600 84 75 20,700 68 50 11,200 37 2 Control 52,000 50 27,300 52 3 Control 23,400 67,500 50 18,400 78 65,000 96 Isotonic sodium phosphate buffer, pH 7.4. and the same buffer diluted with distilled water svere used as the incubation Tnedia.

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134 PART II. BIOSYNTHESIS OF HEMOGLOBIN' The synthesis of heme, however, is not significantly reduced in a medium 50~%o of isotonicity (table II). The differential effect of hypotonicity is con- sistent with the findings that heme synthesis proceeds in non-intact avian erythrocytes3-, and that the incorporation of amino acids into the protein of rabbit reticulocytes ceases if the cells are lysed.7 Influence of Amino Acids and Glucose. Addition of glucose or of a mix- ture of amino acids to the cell suspension results in only slight enhancement of the incorporation of glycine into globin during incubation periods of four hours. With longer periods of incubation, significantly greater incorporation occurs in those preparations to which glucose or amino acids and glucose have been added (Eg. ~ ~ . ~ 4xi04 _ L) - Z- 3x104 _ o Z 2x104 o ,_ 104 - ~_ O +AMINO ACIDS 2 - + GLUCOSE . _ = ~ 105 _ +AMINO ACIDS _ . ~~ ' ~ . 10 .5 4 10 20 1.5 4 10 20 TIME IN HOURS Time in Hours FIG. 1. (left) The influence of amino acids and of glucose on the incorporation of glycine into glol~in in duck erythrocytes. FIG. 2. (right)The effect of time of incubation on the biosynthesis of heme and on the incorporation of glycine into globin in duck erythrocytes. Time of Incubation. The rates of heme synthesis and of incorporation of glycine into globin are most rapid in the initial hours of incubation. With longer incubation both processes are decelerated, but the incorporation of glycine into globin is more markedly slowed. The changes in the rates of the two processes are reflected in the progressive increase in the heme-globin ratios (fig. 2). Temperature of Incubation Median. Both processes are sensitive to temperature, but the sensitivity of heme synthesis is more striking. At 40 C., the synthesis of heme was 100 times more active than at 10 , while the incorporation of glycine into globin was increased only twenty-six times (table III). The Efects of Iron, Cobalt and Lead. Further evidence for the dissocia- tion of heme synthesis and of the incorporation of glycine into globin is pro- vided in the studies on the effects of iron, cobalt and lead or the two pro-

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GLYCINE INTO GLOBIN- LONDON, MORELL AND KASSENAAR 135 TABLE III INFLUENCE OF TEMPERATURE OR SYNTHESIS OF HEME AND INCORPORATION OF GLYCINE INTO GLOBIN Temperature C. Specific activity of glycine (c.p.m./mM) "Eeme/ Globin Ratio" in gIol~.n in heme - 10 800 1~070 1.3 20 3~500 9~800 2.8 30 14,900 48,500 3.2 40 20,600 107,000 5.2 Incubation time four hours. Composition of incubation mixture as described in standard conditions. cesses. Previous studies have demonstrated the inhibitory effects of leader i' and cobalti3 on heme synthesis in duck and chicken erythrocvtes and in bone marrow. The inhibitory effects of these metals can also be demonstrated at an earlier stage of porphyrin synthesis, namely the conversion of delta-amino- levulinic acid to porphobilinogen.~4 The addition of iron on the other hand has been shown to enhance the formation of heme in vitro.5, :0 In table IV the effects of these metals on the incorporation of glycine into Gloria as well as on the synthesis of heme are presented. The enhancement of heme synthesis by iron and the inhibition by lead and cobalt are marked. The incorporation of glycine into globin, however, is unchanged on addition of cobalt or iron and is inhibited to a lesser degree than is heme synthesis by lead. The dissociation of the two mechanisms is reflected in the marked variations that are induced in the heme-globin ratios. Since the addition of iron did not increase the incorporation of glycine into globin of duck erythrocytes, whereas Kruh and Borsook had reported that iron increased the formation of globin in rabbit reticulocytes,S experiments TABLE IV THE EFFECTS OF LEAD, COBALT AND IRON ONT SYNTHESIS OF HEME AND INC~RPCRATION OF GLYCINE INTTO GLOBIN Specific activity of gIycine (c.p.m./mM) Metal Added in Robin per cent of control in heme per cent of control "Heme/Globin Ratio" Control Pb+~- FeCI2 CoCl 15,200 10,800 15,000 1 5,700 71 100 103 50,500 11,600 96,000 2,030 190 4 3.3 1.3 6.4 0.1 Ferrous chloride, lead acetate and cobaltous chloride were added in concentrations of 5 :; 10~ M. 4 ml. of cells were suspended in 8 ml. of previously boiled saline containing metal salts. Glucose, penicillin and streptomycin were added in the usual concentrations. The cells were preincubated with the metals for two hours before substrate was added. After addition of the usual amount of glycine 2-C1-i, the incubation was continued for four hours.

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136 PART II. BIOSYNTHESIS OF HEMOGLOBIN were performed with the blood of rabbits in which reticulocytosis had been induced with acetylphenylhydrazine. The results indicate that the incorpora- tion of glycine into globin in rabbit reticulocytes is enhanced by iron, in con- firmation of the results of Kruh and Borsook, and that the enhancement is very similar in degree to that of heme synthesis (table V). TABLE V INFLUENCE OF IRON ON SYNTHESIS OF HEME AND ON INCORPORATION OF GLYCINE INTO GLOBIN OF RETICULOCYTES OF RABBITS TREATED WITH PHENY~HYDRAZINE Specific activity of glycine ( c.p.m./mM ) "Heme/Globin Sample Ratio" in globin in heme Control 96,800 10,100 1.1 Control 93,400 10,650 1.1 Control 94,200 10,550 1.1 Fe+ ~ 123,600 149,000 1.3 Few ~ 123,500 145,000 1.2 Few ~ 122,600 137,000 1.1 Each sample consisted of 2.5 ml. of cells suspended in 8 ml. of boiled 0.9 per cent NaC1 solu- tion, containing the amino acid mixture, glycine 2-C14 and antibiotics in the same amounts as in the standard incubation mixture. Iron was added as FeCl.~ in the concentration of 40 micro- grams of iron per sample (? x 10~ M). The incubation was carried out for two hours at 37 C. The Endings in rabbit reticulocvres differed from those in normal duck erythrocytes not only in terms of the effects of iron on glycine incorporation into globin, but also in the "heme-globin ratios." "Heme-globin ratios" of 1.1 and 1.2 in rabbit reticulocytes are similar to those previously described.S In the normal duck erythrocytes, however, the "heme-globin ratios" are generally much higher and more variable. To determine whether the "heme- globin ratio" might be closer to unity and whether iron might enhance glycine incorporation into globin in more immature duck erythrocytes, the cells of acetylphenylhydrazine-treated ducks were employed. The results in the more immature erythrocytes confirm the findings observed in the normal duck erythrocytes, namely that the incorporation of glycine into globin is not enhanced by iron, that the synthesis of heme is increased on addition of iron and that the "heme-globin ratio" is usually much greater than unity (table VI ~ . To determine whether incubation of duck erythrocytes in the more natural environment of duck plasma might result in a "heme-globin ratio" closer to unity, experiments were performed in duck plasma, but the "heme- globin ratios" obtained were even higher than those usually found in a phosphate buffer medium. The Effects of N~cleosides. Incubation with purine ribosides has been shown to prolong the viability of stored human and rabbit erythrocytes 17 to enhance the resistance of fresh human erythrocytes to osmotic lysisi8 and to increase the concentration of phosphate esters in human and rabbit erv- throcytes.~9 A Since the mechanism by which the purine ribosides exert these

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GLYCINE INTO GLOBINLONDON, MORELL AND KASSENAAR 137 TABLE VI SYNTHESIS OF HEME AND INCORPORATION OF GLYCINE INTO G~os~N IN ERYTHROCYTES OF NORMAL AND OF ACETY~pHENy~HyDRAz~xE-TREATED DUCKS; EFFECT OF IRON IN ERYTHROCYTES OF ACETY~PHENy~HyDRAz~NE-TREATED DUCKS Expt. No. 1 2 __ ~ ~ Specific Activity of Glycine (c.p.m./mM) Incubation Incubation Time, Duck Medium Hours Erythrocytes in Globin in Heme NaCl 2.5 Normal 48,000 75,000 O.g~o ( boiled ) Acetylphenyl- hydrazine treated a) Control 182,000 406,000 b) Fe++ added 172,000 1,190,000 Isotonic 4 Normal 14,200 48, 5 00 Phosphate Buffer Acetylphenyl- 148, 500 645,000 hydrazine treated ,- "Heme/Globin Ratio" 1.6 2.2 6.9 3.4 4.3 When iron was added, it was in the form of Fecal at a concentration of 5 x 104- M. Standard experimental conditions were observed except as noted. TABLE VII EFFECTS OF PURINE RIBOSIDES AND RELATED COMPOUNDS ON HEME SYNTHESIS AND ON GLYCINE INCORPORATION INTO GLOBIN IN DUCK ERYTHROCYTES AND IN IMMATURE ERYTHRCCYTES OF RABBITS Expt. No. 1 2 3 Erythrocytes Acetylphenyl- hydrazine-treated rabbits Normal Ducks Acetylphenyl- hydrazine-treated ducks Specific Activity of Glycine (amp lmM) Nucleoside "Heme/ Globin Added in Globin in Heme Ratio" Control 10,700 Adenosine, 5 M/ml 7,800 Control 3 3,100 65,000 Adenosine, 10 M/ml 15,300 59,000 Inosine, 10 M/ml 17,700 56,000 Deoxyadenosine, 31,600 59,000 Deoxyguanosine, Cytidine and Thymidine Control 148,500 645,000 Adenosine, 5 M/ml 46,000 296,000 2.0 3.8 3.2 1.9 4.3 6.4

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138 PART II. BIOSYNTHESIS OF HEMOGLOBIN effects is probably the introduction into the erythrocyte of phosphorylated ribose, its metabolism via the he.xose-monophosphate shunt and the Embden- Meyerhof cycle and the formation of high energy phosphate esters, we in- vestigated the effects of the purine ribosides on the incorporation of glycine into globin and on the synthesis of heme. The results of studies in duck erythrocytes and in immature erythrocytes of rabbits are presented in table NIII. In the erythrocytes of normal ducks and of acetylphenylhydrazine- treated ducks, the incorporation of glycine into globin is diminished under the influence of Cosine or adenosine; the degree of inhibition is greater than that noted on heme synthesis. In immature erythrocytes of rabbits the in- hib'2tory influence of adenosine on the incorporation of glycine into globin is observed. The deoxyribosides had no significant effect on either process in normal duck erythrocytes. The incorporation of glycine into the globin of intact erythrocytes repre- sents a composite effect of at least two processes: ~ ~ ~ the uptake of the amino acid by the cell and (2) the incorporation of the amino acid into the globin. The first process has been studied in duck erythrocytes by Christen- sen, Riggs and Ray who showed that normal duel: erythrocytes take up amino acids from a plasma or saline medium against a concentration gradient.") This concentrative activity is considerably less than that which has been observed for guinea pig brain,~'' rat diaphragm'':; or rabbit reticulocytes.~4 Coupled with the lesser concentrative activity of duck erythrocytes for amino acids is a relative insensitivity of the concentrative process to anoxia and to agents such as cyanide, 2,4 dinitrophenol and other metabolic in- hilaitors in high concentrations. a In experiments in normal duck erythrocytes in which the incorporation of glycine into globin has been markedly diminished while heme synthesis has been unimpaired (e.g., in hypotonic media, or under the influence of nucleo- sides) it seems likely that the primary effect is on the mechanism of incorpora- tion of glycine into the protein rather than on uptake of the glycine by the duck erythrocyte. Support for this interpretation may be derived from the finding that the synthesis of heme from glycine is not diminished under these conditions. This finding can be used to support this interpretation on the assumption that the same metabolic pool of glycine is utilized for the syn- thesis of heme and for incorporation of glycine into globin. Further experi- mental work is required to determine whether this assumption of a common metabolic pool is correct. The studies in duck erythrocytes demonstrate that the synthesis of heme and the incorporation of glycine into globin are readily dissociated in vitro by prolonging the time of incubation, by changing the temperature or tonicity of the medium, by the use of metals and by the use of nucleosides. The mechanism for the incorporation of glycine into globin appears more sensi- tive than the synthesis of heme to environmental changes which may induce

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GLYCINE INTO GLOBIN LONDON, MORELL AND KASSENAAR 139 structural disorganization within the erythrocyte. It is, of course, not sur- prising that two processes which are so distinct are differentially affected by these various environmental and metabolic conditions. The Gradings serve to focus attention, however, on the need for determining in various disorders of hemoglobin metabolism the extent to which the synthesis of heme or of globin or of both may be disturbed. These techniques are readily applicable to the study of humeri bone marrows in normal and in disease states. This in vitro system of immature avian or mammalian erythrocytes also provides a suitable tool for the study of the relationship of nucleic acid syn- thesis to hemoglobin formation and of the influences of hormones and of other metabolites on these synthetic processes. Recent work by Mrs. Morell and Dr. Savoie ire our laboratory indicates that rabbit bone marrow in vitro may be used for the study of the temporal relations of heme and globir~ formation. Such studies should complement the investigation in vivo of these mechanisms and of their ir~terrelatior~s.';~~"7 REFERENT CES 1. London, I. NI, Shemin, D., and Rittenberg, D.: a) The i,' ditto synthesis of heme in the human red blood cell of sickle cell anemia, J. Biol. Chem. 171: 797, 1948; b) Synthesis of heme ill vitro by the immature non-nucleated mammalian ery- throcyte, J. Biol. Chem. lS]: 749, 1950. 2. Shemin, D., London, I. M., and Rittenberg, D.: a) The in vitro synthesis of heme from glycine by the nucleated red blood cell, J. Biol. Chem. 171: 799, 1948; b) The synthesis of protoporphyrin in vitro by red blood cells of the duel;, l. Biol. Chem. 181: 757, 1950. 3. Shemin, D., and Kumin, S.: Alpha ketoglutarate-succinate reaction; the forma- tion of a succinyl intermediate for succinate, Federation Proc. 11: 285, 1952. 4. London, I. M., and Yamasaki, M.: Heme synthesis in non-intact mammalian and avian erythrocytes, Federation Bloc. 11: 250, 1952. 5. Dresel, E. I. B., and Falk, l. S.: a) Haem and porphyrin formation from delta- aminolevulinic acid and from porphobilinogen in haemolysed chicken erythro- cytes, Biochem. J. 61: 80, 1956; b) Haem and porphyrin formation in intact chicken erythrocytes, Biochem. J. 63: 72, 1956. 6. Allfrey, V., and Mirsky, A. E.: The incorporation of Ni5-glycine by avian ery- throcytes and reticulocytes in Citron J. Gen. Physiol. 35: 841, 1952. 7. Borsook, H., Deasy, C. L., Haagen-Smit, A. J., Keighley, G., and Lowy, P. H.: Incorporation in Vitro of labeled amino acids into proteins of rabbit reticu- locytes, J. Biol. Chem. 196: 669, 1952. 8. Krnh, J., and Borsook, H.: Hemoglobin synthesis in rabbit reticulocytes in Vitro, J. Biol. Chem. 220: 90 5, 19 5 6. 9. Anson, M. L., and Mirsky, A. E.: Protein coagulation and its reversal. The prep- aration of insoluble Robin, soluble globin and heme, J. Gen. Physiol. 11: 469, 1930. 10. Kassenaar, A., Morell, H., and London, I. M.: The incorporation of glycine into globin and the synthesis of heme in vitro in duck erythrocytes, J. Biol. Chem. (Ir1 Press) 11. Eriksen, L. E.: Lead intoxication: 1. The effect of lead on the in vitro biosyn-