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HEMOGLOBIN SYNTHESIS IN VITRO IN RABBIT RETICULC)CYTES~ HENRY FORSOOK Rabbit reticulocytes in vitro rapidly incorporate labeled amino acids into their proteins.) Nearly all the trichloroacetic acid precipitable material is hemoglobin. In experiments with labeled glycine, histidine, leucine or lysine the results were substantially the same whether the incorporation was measured in the mixture of proteins precipitated by trichloroacetic acid or in the iso- lated hemoglobin. The hemoglobin, as measured by the method of DraLkin and Austin,- in the reticulocytes as prepared by us, is about 1 j per cent, and constitutes nearly all of the dry weight of the material precipitated by tri- chloroacetic acid. These facts and the results of experiments in which the hemoglobin was isolated,3 warrant referring to amino acid incorporation in the proteins of rabbit reticulocytes as protein or hemoglobin synthesis. In the first experiments it was found that amino acid incorporation into the proteins of the reticulocytes was accelerated by the plasma of every mammal investigated and also by extracts of normal erythrocytes, rabbit reticulocytes, liver, spleen and yeast. We undertook the identification of the accelerating factors. The total accelerating effect, potential as well as actual, of plasma and tissue extracts has been accounted for by known substances they contain. Production of Reticalocytosis. Reticulocytosis was produced in adult rabbits by a modification of the method of London et al.4 One milliliter of a neutralized 2.5 per cent aqueous solution of phenylLydrazine hydrochloride divas injected subcutaneously each day for a week; over 90 per cent of the circulating red cells were then reticulocytes. Labeled amino acids. Methyler~e-labeled C~'~-glycine was obtained from Tracerlab, Inc.; as used, its specific activity was 5.5 x 106 c.p.m. per mmole. L-histidine, L-leucine and L-lysine were prepared labeled with C2'' in their carboxyl groups;' 5 their specific activities were respectively, 2.21, 2.29 and 1.56 ~ 106 c.p.m. per mmole. Their initial concentration in the reaction mix- ture was always 0.001 M. Incubation Procedure. The incubation was carried out in 20 ml. beakers in the DuLnoff apparatus6 under 95 per cent 02 arid 5 per cent COP at 37.5 C. for lengths of time indicated below. with a rocking rate of 100 cycles Per O , These studies were aided by a contract between the Atomic Energy Commission and the Division of Biology, California Institute of Technology. They were also sup- ported by a research grant from the National Institutes of Health, United States Public Health Service, and by a grant-in-aid from the American Cancer Society upon recommendation of the Committee on Growth of the National Research Council. The following were collaborators in the work described here: Drs. A. Abrams C. L. Deasy, E. H. Fischer, G. Keighley, J. Kruh, and R. H. Lowy. 111

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112 PART II. BIOSYNTHESIS OF HEMOGLOBIN minute. Each beaker contained 0.5 ml. packed reticulocytes in 4 ml of re- action mixture. The solvent was either Krebs-Henseleit solution; or the latter solution with omission of calcium and phosphate salts (see below the reasons for the omission) as indicated. All the glassware and all the solutions except the amino acid mixture were sterilized in the autoclave. Since repeated autoclaving was found to destroy phenylalanine, the amino acid mixture was boiled once and then kept in the deep freezer in small flasks with just enough, usually 30 ml., for one experi- ment per flask. Preparation of Mixed! Proteins. At the end of the incubation the contents of each beaker were added to 80 ml. of water and the reticulocyte protein precipitated with 20 ml. of 35 per cent trichloroacetic acid. If the reaction mixture contained plasma or some other proteins the cells were first separated by centrifugation and washed twice with saline, before the trichloroacetic acid precipitation. After the precipitated protein had settled, leaving a clear su- pernatant solution, usually in about three hours, the supernatant solution was decanted off so as to leave a well drained paste of precipitated protein, which was then dissolved in 1 N NaOH and then reprecipitated by 100 ml. of 7 per cent trichloroacetic acid. After it had settled well, the precipitated protein was transferred to a centrifuge tube, in which it was washed twice with 7 per cent trichloroacetic acid, twice with a mixture of equal volumes of acetone and ether, once with acetone, twice with ether, and then dried at 85~. Preparation of Hemoglobin. The hemoglobin divas isolated and purified by a modification of the method of Roche et al.S~~ Equivalent amounts of KH~PO4 and K2HPO4 were added to give 2.9 M total phosphate. All the plasma and cellular proteins were salted out, whereas rabbit hemoglobin, which is extremely soluble, remained in solution. After standing overnight at room temperature, the salted-out proteins were removed by filtration. The hemoglobin was then salted out by further addition to the filtrate of equiva- lent amounts of the two salts to give 3.5 M phosphate. The hemoglobin was collected by filtration on a Buchner funnel and washed on a filter with 3.5 M phosphate solution. The washed hemoglobin was then dissolved in about 50 ml. Of water and dialyzed at 4 for 3 days against repeated changes of water total volume ~ 0 liters ~ . Preparation of Heme. The heme was isolated from the hemoglobin by an adaptation of the method of Anson and ~lirsky.~i Ten volumes of 1 per cent HC1 in acetone were added to the hemoglobin solution. The globin, which precipitated, was removed by filtration, an equal volume of water was added to the filtrate, and the diluted acetone solution was evaporated under a lamp. After a few hours the heme precipitated; it was washed by decantation four times with water, dried overnight at 100 C, dissolved in 0.5 ml. pyridine, and filtered onto a circle of lens paper that fitted exactly in a circular alum- inum cup, 19 mm. in diameter, and dried under a lamp. With 8 to 15 ma.

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HEMOGLOBIN SYNTHESIS IN RETICULOCYTESBORSOOK 1~ 3 of heme, a uniform spread was obtained for the measurement of the radio- activity. Preparation of Globin. The globin which had been filtered from the heme was washed on the filter with 1 per cent HC1 in acetone until the washings were colorless. It was then suspended in 80 ml. of 7 per cent tri- chloroacetic acid. The remainder of the procedure whereby it divas obtained in a dry state for the measurement of its radioactivity was identical to that of the mixed proteins described above. Measurement of Radioactivity. The material --'lose radioactivity divas to be measured was spread uniformly on an aluminum plate 19 mm. in diameter and counted in a Geiger-Muller end window counter. An empirical self- absorption curve was used to correct for the thickness of the sample. Calculations of Heme. Heme-pyridine hemochromogen contains 2 resi- dues of pyridine per heme;i2 8 molecules of glycine enter into the synthesis of 1 of home.: On this basis the number of millimoles of heme synthesized per I/4 molecule of hemoglobin (or per mole of heme attached to hemoglobin' isolated is given by ~ (counts per minute per ma. of heme-pyridine/(counts per minute per millimole of labeled glycine ~ 8) ~ Y 774 ~ 103. Incorporation of Amino Acids into Globin. In order to compare the rates of incorporation of four different amino acids into globin it is neces- sary to take into account the amounts of these amino acids in the globin. There are no reliable analytical data on rabbit hemoglobin. Accordingly, we have used the following data of Schroeder et aI.14 on human hemoglobin expressed as residues per molecule of hemoglobin: glycine 43.1, histidine 36.3, leucine 76.0, and lysine 44.0. Tristram i~ gives the following (as residues per molecule of hemoglobin) for horse hemoglobin: glycine 48, histidine 36, leucine 75, and lysine 38.0. In view of the similar amino acid composition of human and horse hemoglobins it seems likely that the values obtained by Schroeder et al. for human hemoglobin can be used for rabbit hemoglobin for purposes such as ours, without significant error. The number of millimoles of labeled amino acid incorporated into hemo- globin per residue of that amino acid in the protein let glycine be an example- is given by ~ (counts per minute per ma. of globin/ (counts per minute per millimole of glycine x 43.1 ) ~ x 6.6 ~ 104. Results. Table I is a summary of the main factors we have found which accelerate protein synthesis in vitro in rabbit reticulocytes. Not included are fructose-amino acids whose effects are referred to below in discussing the effect of iron. It is seen that iron and glucose, separately or together, had little accelerating effect. The amino acid mixture alone caused an increase of 70 per cent; amino acids and iron, but not amino acids and glucose, acted synergistically; glucose acted synergistically when added with amino acids and iron. Transferrin, added with amino acids, iron and glucose increased the rate still further. These results indicate that for a high rate of synthesis the

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114 PART II. BIOSYNTHESIS OF HEMOGLOBIN TABLE I FACTORS ACCELERATING PROTEIN SYNTHESIS IN RABBITT RETICULOCYTES IN VITRO. Amino acid mixture Iron Glucose Trans- Cam. 1 ma. per 1errin per ml. ml. 50 Ham. per ml. _ _ + _ _ ~ + _ + _ _ _ ~ + _ _ + _ + _ + + _ + - Rate of protein synthesis 100 100 110 111 170 351 170 497 597 TABLE I.The composition of the amino acid mixture is given in the text. The iron was in the form of ferrous ammonium sulfate. The transferrin was a crystalline metal-free preparation. constituents of hemoglobin, i.e. amino acids and iron, and an energy source, i.e. glucose, must be available in adequate amounts. The result with trans- ferrin points to the usefulness of an iron-chelating agent even though it may not be strictly necessary. Amino Acid Mixture The amino acid mixture used had the following composition, expressed as Am. per ml. of reaction mixture: L-alanine 45, L-arginine 21, L-aspartic acid 95, L-cysteine 12.5, glycine 100, L-glutamine 70, L-histidine 90, L-hydroxyproline 37.5, L-isoleucine 10, L-lysine 65. L- methionine 12.5, L-phenylalanine 65, L-proline 40, DL-serine 90, L-threonine 50, L-tryptophane 15, L-tyrosine 37.5, and L-valine 90. L-leucine was pro- vided in the carboxyl-C7; form and at a concentration of 10-3 M (131 Am. per ml.~. Only nine of the above nineteen amino acids were found to be in any degree limiting during a four-hour experiment. These, except leucine, are given in table II. In other experiments) it had been found that leucine is severely limiting. Three points may be made regarding results such as those in table II. Amino acids vary in the degree to which they are limiting; histidine is the most limiting. In a reaction mixture from which one of the limiting amino acids is withheld, the rate is at a characteristic suboptimal level from the beginning and persists so. Throughout the four hours glutamine had an accelerating effect, whereas added glutamic acid had no effect. In separate experiments it was found that C~'~-labeled glutamic acid Was not incorporated at all, whereas Ct4-glutamine was extensively incorporated. Evidently gluta- mic acid is unable to penetrate into the reticulocyte whereas glutamine can.

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HEMOGLOBIN SYNTHESIS IN RETICULOCYTES BORSOOK TABLE II EFFECT OF AMINO ACID COMPOSITION RESULTS EXPRESSED AS PER CENT OF THAT IN SALINE ALONE WITHOUT ADDED A MINO ACIDS. 11; Hours Amino Acid Composition Complete " without histidine ,, " valine ,, ,. .. ,. .. ,, .. ,, ,, ,, 1 447 94 185 176 215 235 257 262 304 2 3 450 489 87 94 170 181 183 181 227 220 229 237 267 264 280 295 339 353 4 475 96 187 200 228 222 285 297 366 TABLE II. The complete reaction mixture contained the amino acid mixture de- scribed in the text, 5 hum. per ml. of iron as ferrous ammonium sulfate and 1 ma. per ml. of glucose. It did not contain transferrin. It has been observed that glutamic acid penetrates the cells of liver slices slowly. The limiting amino acids in rabbit reticulocytes include both those which are dispensable and indispensable. Isoleucine, methionine and threonine were not limiting. Although reticulocytes synthesize serine from glycinei and pre- sumably also synthesize glutamic acid, they do not make enough for a maximum rate of protein synthesis. These findings illustrate that the amino acid re- quirements of different tissues may be quite different from that of the animal as a whole. TABLE III EFFECT OF PARTIAL FIISTIDINE DEFICIENCY. RESULTS EXPRESSED AS PER CENT OF THAT IN COMPLETE REACTION MIXTURE. Amount of histidine added to otherwise complete reaction mixture gm./ml. molal O O 9.25 l8.S 27.75 37.0 46.25 74.0 Rate in successive hours 0.575 x 10-4 1.15 " " 1.72 " " 2.30 2.87 5.75 ,. .. ,. .. ,. .. Q-1 1 1-2 2-3 18 27 30 33 77 39 98 93 98 88 97 98 100 100 23 81 104 100 100 100 100 3-4 23 24 25 47 80 102 100

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116 PART II. BIOSYNTHESIS OF HEMOGLOBIN Tables III, IV and V show in more detail how the quality of the amino acid mixture may determine the rate of protein synthesis. With suboptimal concentrations of the limiting amino acids the rate is at first nearly maximal, then declines, but may remain well above the minimal rate. Of course all the amino acids in hemoglobin are needed for its synthesis. The amino acid requirement in the reaction medium for a maximum rate of synthesis is inversely related to the amino acid content of the cells. Accord- ingly, in the case of reticulocytes of a different species, e.g. avian cells, the amino acid mixture required to be added to the reaction mixture may be different from that necessary for rabbit reticulocytes. The literature on the relation between amino acids and blood formation TABLE IV EFFECT OF PARTIAL PHENYLALANINE DEFICIENCY. RESULTS EXPRESSED AS PER CENT OF THAT IN COMPLETE REACTION MIXTURE. Amount of phenylalanine added to otherwise com- plete reaction mixture. Am. / ml. o 6.5 13.0 19.5 26.0 32.5 65.0 Rate in . , successive nours 0-1 molal o 0.39 X 10 - 4 0.78 " " 1.18 " " 1.57 1.96 3.93 " " ,. .. ,. .. 35 88 90 92 93 94 100 -2 2-3 3-4 37 39 66 62 90 87 93 105 92 100 93 102 100 100 38 61 80 98 102 101 100 TABLE V EFFECT OF PARTIAL VALINE DEFICIENCY. RESULTS EXPRESSED AS PER CENT OF THAT IN COMPLETE REACTION MIXTURE. Amount of valine added to otherwise complete reaction mixture ,ugm./ml. o 9.25 18.5 27.75 37.0 46.25 92.5 Rate in successive hours 0-1 molal o 0.78 X 10 - 4 1.57 " " 2.3 6 I' 7, 3.15 3.94 7.89 ,. .. ,. .. ,. .. 81 98 100 95 95 100 1-2 1 2-3 1 3-4 29 61 85 97 99 99 100 50 71 84 92 94 100 32 53 73 86 98 97 100

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HEMOGLOBIN SYNTHESIS IN RETICULOCYTES BORSOOK 117 in animals is extensive, i7~39 but the results reported are unsystematic and some are conflicting. Nevertheless, the evidence from in Gino experiments tend to extend the significance of the relationship found between labeled amino acid incorporation into reticulocyte proteins and hemoglobin synthesis. For example Sebrell37 found that omission of some of the essential amino acids from the diet of rats made anemic by bleeding handicapped red cell regeneration more than the omission of others. The amino acids found to be most necessary for red cell and hemoglobin regeneration, stated in order of their effectiveness, were histidine, valine, lysine, phenylalanine, and leucine. The list agrees surprisingly well with the amino acids accelerating incorpora- tion of labeled amino acids into rabbit reticulocyte proteins. Nizet and Robscheit-Robbins3S found that dog reticulocytes did riot mature so quickly in vitro in the blood of dogs with hemorrhagic anemia and hypo- proteinemia as in normal blood, unless a mixture of the ten essential amino acids and glycine was added to the anemic blood. Variations from animal to animal were too great for any conclusions to be drawn regarding differences among the individual amino acids. Orten and Orten 3- studied the effects on hemoglobin regeneration of adding individual amino acids to the diet of young rats previously made anemic by a low protein diet. They concluded that there was no "key" amino acid; however, some amino acids appeared to be more effective than others in their experiments. Art apparent discrepancy had existed between feeding experiments on the one hand, and those on the incorporation of labeled amino acids on the other. In feeding experiments it was found that an indispensable amino acid is in- ~ r 1 effective for growth, for recovery from protein depletion, or for maintenance, unless it is fed or injected within a few hours of other necessary amino acids. The same applies to the carbohydrate in a meal with respect to the caloric re- quirement for growth or nitrogen balance.3S~~5i The interpretation placed on these Endings has been that all the amino acids must be present (presumably in the blood) at concentrations greater than the fasting levels for protein synthesis to occur. Against this interpretation stand the observations that a single amino acid, whether dispensable or indispensable, when injected or fed in labeled form can be seen to be extensively incorporated into the proteins of the animal in a few minutes. Extensive incorporation occurs whether the animal is normally fed or fasting. All the above (and similar) observations are in accord with the following interpretation. An animal's protein is, viewed in toto, always in a dynamic state. Growth, balance or loss in weight connote respectively more, equal, or less synthesis than breakdown of protein. For maximum synthesis all the necessary amino acids and an adequate source of energy Carbohydrate) need to be present at the same time (tables I and XIII). If orate amino acid is present in suboptimal amount the rate of protein synthesis is suboptimal in degree according to the degree of the deficiency. There is never a complete

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llg PART II. BIOSYNTHESIS OF HEMOGLOBIN cessation of protein synthesis; hence under all conditions all amino acids are incorporated, and the rates of incorporation of all are affected to the same extent. Hence when in vivo an indispensable amino acid is withheld or given much later than the main meal, protein synthesis is too slow for growth or maintenance. Feeding the ~-vitheld amino acid later does not correct the deficiency because amino acids, whether inj ected or fed, are too rapidly cleared from the blood. Correspondingly a single amino acid, whether in- jected or fed, will be extensively incorporated because protein synthesis never stops in Volvo. In this sense the results in tables II - ~ and XIII are analogues of experiments in which animals are maintained on suboptimal amounts of indispensable amino acids (or incomplete proteins) or inadequate calories. Iron. The effect of the addition of iron to the reaction mixture is shown in table VI. F`eSO~ (NH~), SO4.6H`,O was used mostly; ferrous chloride divas equally effective but less convenient; ferric chloride was almost as effective. To obtain consistent and maximal effects it was necessary to add the iron to the reaction mixture after the ret~culocytes, as the last ingredient before incubation. The reaction mixture divas somewhat alkaline (pH about 8 ~ before the reticulocytes there added and before being placed under 95 per cent oxygen and 5 per cent carbon dioxide, and the iron may be pre- _ipitated when added at this point. FABLE VI EFFECT OF IRON Reaction mixture except for iron and transferrin Saline alone Complete 77 Concentration of iron molal x 10 A 0.35 0.89 1.79 (1 ~gm./m1.) 3.58 8.95 17.90 Leucine incorporated OCR for page 111
HEMOGLOBIN SYNTHESIS I)l RETICULOCYTES BORSOOK 119 TABLE VII I NCREASE INT IRON BOUND AS HEMCGLOBIN WITH INCREASING AMOUNTS OF IRON- ADDED TO THE REACTIONS MEDIUM. Iron added to reaction 1' n~ealum concen- tratlon: ham. per ml. o 1.0 2.5 5.0 10.0 20.0 40.0 Total iron bound as h~moglob.n clueing periods of 1 to 4 hours. ,umoles >; 10 total: Moles x 10 o 0.17 0.44 1 hr. .092 0.16 0.17 0.89 0.18 1.79 0.17 3.58 0.18 7.16 0.18 2 furs 0.14 0.21 0.25 0.28 0.30 0.30 0.30 Increase in bound iron in 4 furs. consequent on addition of iron ,umoles x 10 4 furs. 0.16 0.24 0.30 0.34 0.38 0.38 0.38 .08 .14 .18 .22 .22 .22 oracles of leucine per gram of protein corresponds per beaker to an in- creased incorporation of about 0.5 x 10-1 Moles of iron into hemoglobin. To obtain this increase the addition of 1.6 ~ 10-i Moles of iron per beaker alas needed. Evidently there is very little iron in reticulocytes available for hemoglobin synthesis. The data indicate that some of the iron added to the reaction medium - was rendered unavailable. This is the interpretation we have placed on data such as those in table VII. It is seen that during the first hour the addition of a very small amount of iron, between 0.017 and 0.044 Insoles, had a pearl', maximal effect; whereas over a four-hour period between 0.089 and 0.179 Moles were required for a maximum increase in hemoglobin iron of only 0.022 ~moles. The sensitivity of rabbit reticulocytes in vitro to such low concentrations of iron raises the question whether or not iron is necessary for hemoglobin synthesis in addition to its participation in the structure of heme. Speakers at this symposium have stated that iron is needed by at least one of the enzymes required for the synthesis of heme at a stage prior to addition of iron to protoporphyrin. We have no information on whether iron may be needed by the enzymes involved in globin synthesis. The addition of cobalt ~ as CoCl~ ~ also accelerated protein synthesis in. reticulocytes, table VIII, but less than did the addition of iron. The minimum amount giving a maximum effect was of the same order of magnitude in both, 2-5xI0-,:\~. Table IX shows that when cobalt was added with iron at comparable con- centrations, only the accelerating effect of the iron was obtained.

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120 PART II. BIOSYNTHESIS OF HEMOGLOBIN TABLE VIII RELATIVE ACCELERATING EFFECTS OF COBALT AND OF IRON. RESULTS EXPRESSED AS PER CENT OF VALUE WITHOUT EITHER METAL. Concentration of added metal molal 1 x 10 - 4 5 x 10 - 5 2 X " 1 x " 5 X 10 Cobalt 151 156 113 110 100 Iron 229 223 ~ 1 ~ lg8 154 * The cobalt was added as CoCl.,. TABLE IX NON-ADDITIVITY OF THE ACCELERATING EFFECTS OF COBALT AND IRON. RESULTS EXPRESSED AS PER CENT OF VALUE WITHOUT EITHER METAL. Concentration of iron molal 1 X 10 5 x " 2 x " 1 x " 5 X 10 - 6 Leucine incorporated when, in addition to iron, cobalt was added at a 2 x 10 M 1 ~ 10~ M 229 223 218 222 200 183 1Si 141 212 188 154 5 x 10c M 208 189 141 Experiments were carried out also with aluminum, manganese, molybdenum and zinc at concentrations of 0.2-1.0 x 1O-4 M with and without added iron, table X. They were all slightly inhibitory when the iron was 1O-4 M; at lower concentrations of iron they had no effect. No transferrin was used in any of the above experiments with metals. 1 S' 40 ~ 30 z ~ 20 z 0 10 c, / ~ ~ rat 2 3 4 HOU R S FIG. 1. Effect of medium on heme syn- thesis in rabbit reticulocytes. /\ Plasma ~ amino acids + iron + glucose O - Non-protein plasma filtrate dialyzed + amino acids + iron + Amino acids + iron ~ glucose Blank

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HEMOGLOBIN SYNTHESIS IN RETICULOCYTES BORSOOK 121 TABLE X EFFECT OF METALS RESULTS EXPRESSED AS PER CENT OF BLANK Metal added None Aluminum Cobalt Copper Manganese Molybdenum Zinc Additional Iron molar concentration I mol21 a ~1 0.2 X 10 - 4 1.0 '' ,' 0.2 1.0 0.2 1.0 0.2 1.0 0.2 1.0 0.2 1.0 ,' ~7 100 100 102 123 239 109 116 116 116 97 102 109 93 0.9 x 105 1 0.2 X 104~ 1 0.9 X 10 186 186 206 174 283 316 300 293 237 293 TABLE XI COMPARISON OF THE ACCELERATING EFFECTS OF PLAS.MA (RABBIT) AND OF TRANSFERRIN. RESULTS EXPRESSED AS PER CENT OF VALUE IN- OTHERWISE COMPLETE REACTION MIXTURE. Protein added None Plasma ,, i, ,, ,' Transferrin ,' ,' ,, ,, Rabbitt serum albumin Bovine " " Human ~~ i, . Transferrin Amount concentration added added to reaction mixture . per ml. reaction Bum. per ml. mixture O 0.05 ml. 125 (estimated) 0.025 " 62.5 '' 0.005 " 12.5 " 0.001 " 2.5 " 0.0005 " 1.25 " 200 60 20 4 2 500 ,t4gm. O 500 Bum. 0 500 Bum. 0 Incorporation of leucine 100 141 135 125 118 100 141 134 132 110 100 97 98 97

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122 PART II. BIOSYNTHESIS OF HEMOGLOBIN Transferrin causes an increase above that obtained in a reaction mixture optimal with respect to amino acids, glucose and iron. A similar effect had been obtained with whole plasma (rabbit and human) (fig. 1~; in twenty- three experiments rabbit plasma caused an increase of 25 +12 per cent over that in an otherwise optimal reaction mixture. The active principle was not dialyzable, and disappeared on boiling; fractionation suggested that it might be transferrin. As table XI show-e this surmise appears to have been correct. The estimate in table XI of the transferrin in the plasma added is based ore DraLkin's value '~ of 0.25 am. per 100 ml. plasma. The transferrin used was kindly provided by Dr. l. L. Oncley of Harvard University.* The specificity of transferrin is attested to further by the absence of any effect of rabbit, bovine or human serum albumin. Presumably the eFectiveness of transferrin comes from its capacity to chelate and so transfer iron. It is surmised that the accelerating effects of fructose- amino acids and citrate are for the same reason, table XII. The maximum accelerating effect is obtained with about 10-6 M transferrin and 5 x 10-4 NI fructose- or tagatose-amino acids or citrate. The different fructose- and tagatose amino acids are Amadori rearrange- TABLE XII ACCELER.~\TING EFFECTS OF FRUCTOSE- AND TAGATOSE-AMINO ACIDS, AND OF CITRATE. RESULTS EXPRESSED AS PER CENT OF VA! UE LIT OTHERWISE COMPLETE REACTION MIXTUR E. Substance i Fructose-Alanine " -Arginine -Aspartic acid " - Glutamic " -Glycine -Histidine . -Leuclne -Lysine -Phenylalanine -Serine i, ,, ,' ,, ', -Threonine ,? -Valine l a~,atose-Alanine Citrate ', -Glyci ne 120 135 125 113 118 130 123 129 125 125 121 120 127 128 108 i The initial concentration of all of the above substances was 5 ~; 10I- M. Iron Concentration 0~5 ,u~m/ml | 5~0 ,ugm/ml. 105 102 104 :> It was prepared from human plasma, crystalline, metal-free, and consisted of 96 per cent 0-1 metal-binding protein, 3 per cent p-2 and a- and 1 per cent of oc-globulins.

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HEMOGLOBIN SYNTHESIS IN RETICULOCYTES BORSOOK 123 ment products of N-glycosyl-amino acids,53~~;' made respectively from glucose and galactose; glucose and mannose give the same product. It is seen that the effect was approximately the same with all the sugar-amino acid compounds, and that they are relatively more effective at suboptimal concentrations of iron, 0.5 mu gm/ml, than at optimal concentrations, ~ 2 ~gmiml. Transferrin on the other hand is more effective at optimal iron concentrations. Citrate was somewhat less effective than the sugar-amino acid compounds. Table XIII shows the accelerating effect of glucose added to the reaction mixture. It is seen that the effect was little in the first hour of incubation and became progressively greater, presumably as the carbohydrate initially in the reticulocytes was consumed. TABLE XIII EFFECT OF GLUCOSE. Incorporation c/m. ma. Differential effect of glucose c/m. my. ~ i Time: Hours 1 Saline + glucose ~ amino acids + glucose + amino acids Per cent of total . . Incorporation 3.32 3.12 14.59 15.01 2 3 4 1 2 4.75 6.38 7.43 5.03 6.89 7.72 -0.20 0.38 22.94 25.78 26.01 26.15 34.05 36.84 0.42 3.21 2.7 1 12.2 3 0.51 8.27 4 0.29 10.83 24.2 1 29.3 TABLE XIII. The added glucose was 1 ma. per ml.; the amino acids were the complete mixture described above, the iron was 5 Am. per ml. TABLE XIV EFFECTS OF SODIUM AND OF POTASSIUM. Nature of saline solution Leucine incorporated ~moles/gm. protein Krebs-Henseleit saline solution All-sodium " " All-potassium " " ~/2 sodium + ~/2 potassium " ', 20.1 19.8 10.9 19.0 TABLE XIV. "All-sodium" and "all-potassium" saline solutions: the potassium and sodium salts of the Krebs-Henseleit solution were replaced by the corresponding sodium and potassium salts respectively.

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124 PART II. BIOSVIN'THESIS OF HEMOGLOBIN TABLE XV EFFECTS OF MAGNESIUM AND OF PHOSPHATE. Leucine Mg. PO4 incorporated molal molal ~moles/gm. protein 0 0 18.2 0.008 0 18.6 0.0016 0 18.6 0 0.008 1 1.0 0 0.016 17.6 0.008 0.008 16.8 0.008 0.0016 18.6 0.0016 0.008 15.2 0.0016 0.0016 17.4 TABLE XV. The basic saline solution was the Krebs-Henseleit mixture from which the calcium, magnesium and phosphate salts were omitted. TABLE XVI EFFECTS OF CALCIUM AND OF CITRATE. Leucine CaCl~, Citrate incorporated molal molal ~moles/gm. protein 0 0 18.4 2.8 x 10 - 3 0 11.6 0 0.25 x 10 - :, 19.4 0 1.0 " 19.1 2.8 x 10 - 3 0.25 " 17.3 2.8 " 1.0 " 19.7 TABLE XVI. The basic saline solution was as in Table XIII. Tables XIV, XV, and XVI show some results of varying the electrolyte composition of the saline solution in which the cells were incubated. An all- potassium, but not an all-sodium saline solution was inhibitory; the inhibition by potassium was neutralized by sodium, table XIV; phosphate was inhibitory arid its inhibitory effect was neutralized by magnesium, table XV; calcium was inhibitory, its inhibitory effect was relieved by citrate, table XVI. After these observations the Krebs-Henseleit solution we had used heretofore was modified by omission of the calcium and phosphate salts. Tables XVII, XVIII and XIX show the effects of a variety of inhibitors at a range of concentration. The most unexpected result was that lead (as lead acetate) was the most powerful inhibitor found; it was much more in- hibitory than antimony, gold or mercury, which last were about the same. Among the antibiotics tested, aureomycin alas the most inhibitory and chloram-

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HEMOGLOBIN SYNTHESIS IN RETICULOCYTESBORSOOK 125 TABLE XVII EFFECT OF SO,ME OXIDATION, PHOSPHORYLATION AND OTHER INHIBITORS. RESULTS EXPRESSED AS PER CENT INHIBITION. Concentration: moIal 0 - 3 Arsenate Arsenite 2,4-D initrophenol ~ -Ethylmaleimide Diisopropylfluorophosphate PCMB Hg-Phenylsulfonate Lead-acetate 90 99 97 6 o 95 95 98 0 35 s 96 o o 9 21 96 0~ ~ 10 4 2 1 1 3 o o o o 18 24 o o o 2 79 o TABLE ~VIII EFFECT OF SOME INHIBITORY METALS. RESULTS EXPRESSED AS PER CENT INHIBITION. Concentration: molal lo - 3 Ammonium Alum Potassium Alum Antimony Potassium Tartrate Cupric Chloride Gold Lead Acetate Mercuric Acetate 17 10 98 16 96 98 100 2 4 93 o 84 96 92 10~ 1 10~ 1 10 - '3 1- o 4 5 o 2 79 5 o o o o o 18 o TABLE XIX EFFECTS OF SQME AMINO ACID, PURINE AND PYRIMIDINE ANALOGS AND ANTIBrOTICS. RESULTS EXPRESSED AS PER CENT INHIBITION. (~,one~?n t.rs~ tinn ~ rn r~~n ~ Benzimidazole 2,6-Diaminopurine sulfate 8-Azaguanine 4-Phthalimido-2,6- dimethylpyrimidine Chloramphenicol Aureomycin 19 ~8 12 42 93 71 2 13 o o 13 56 o o o o 6 45 2.5 x 10~ 1 x 10 o o o o 12 o o o o o o

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126 PART II. BIOSYNTHESIS OF HEMOGLOBIN phenicol next; bacitracin, penicillin G and streptomycin caused less than 10 per cent inhibition at 5 x 10-3 M. The following amino acid, purine and pyrimidine analogs caused less than 10 per cent inhibition at 5 x 10-3 M: o- fluorophenylalanine, 3-fluoro-L-tyrosine, p-2-thienylalanine, 4-amino-5-imid- azolecarboxamide, isoguanine sulfate, 6-mercaptopurine, 6-aminouracil, 5- bromouracil, 4,6-diLydroxypyrimidine, 2-thiocytosine. TABLE XX COMPARISON OF RATES OF HEME SYNTHESIS AND CONCURRENT INCORPORATION OF GLYCINE INTO GLOBIN.3 Duration (1) furs. 1 2 4 Number of Heme Glycine experiments Synthes s incorporated (2) (3) (4) mM/M heme mM/gly residue In global 2 1.67-2.87 2.19- 2.80 21 2.48-7.51 2.71- 6.30 16 2.53-9.36 2.76-10.33 ( 3 ) (5) 1.14 + 0.24 0.98 + 0.15 1.06 + 0.14 In rabbit reticulocytes in vitro the rates of synthesis of heme and of glycine incorporation into globin per glycine residue were equal, table XX. This was observed with rates of hemoglobin synthesis which were made to vary widely by variatiorls in the iron fructose-amino acids in the reaction mixture. This result would not have occurred if there had been a pool of significant size of unlabeled intermediates of either heme or of globin, or of free heme or globinO The rates of synthesis of the two parts of hemoglobin under our experimental conditions, i.e. rabbit reticulocytes in vitro over periods not greater than hours, must have been nearly the same. This conclusion requires that the relative rates of incorporation of different amino acids into globin be the same. This was tested. The procedure was to add labeled glycine, histidine, leucine or lysine to different aliquots of cells containing otherwise the same reaction mixture, and then to incubate all the aliquots concurrently under identical conditions. Different conditions of stimulation were used in a number of different experiments. The results obtained, summarized ire table ~XI, were that per mole of amino acid in TABLE XXI RELATIVE RATES OF INCORPORATION IN GLOBIN OF GLYCINE, HISTIDINE, LEUCINE AND LYSINE.:3 His/Gly Leu/Gly Lys I Gly His /Leu 1.09 + 0.35 1.01 ~ 0.27 0.84 + 0.18 0.98 ~ 0.05

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HEMOGLOBIN SYNTHESIS IN RETICULOCYTES BORSOOK 127 g;lobin, glycine, histidine, leucine, and lysine were incorporated at the same rate. All the deviations from 1 ire the different sets of ratios are within the experimental error and the uncertainties of the correction factor applied to the glycine incorporation for the change ire specific activity of the glycine within the cells. The foregoing observations indicate that in rabbit reticulocytes in vitro the rates of synthesis of heme and of globin are somehow regulated so that both proceed at or nearly at the same rate. The two processes are so different that large differences might have been expected. The above observations notwith- standing, with any marked pathological changes induced within the cells, it may be expected that the regulation would be broken down. Radiations and starvations appear to have such an effect; heme synthesis appears to be the more sensitive of the two processes. The two processes are more easily dislocated in avian than in rabbit cells.59 In disease (human) where there is interest in changes in the blood, the hemoglobin concentration is noted with such other hematological data as red cell count and hematocrit value. It might be that in some diseases the dis- location of heme and of globin synthesis is more pathognomonic than a possible anemia, i.e. suppression of the synthesis of both parts of hemoglobin or abnormally rapid destruction of red cells. REFERENCES 1. Borsook, H., Deasy, C. L., Haagen-Smit, A. J., Keighley, G., and Lo by, P. H.: Incorporation in vitro of labeled amino acids into proteins of rabbit reticulo- cytes, J. Biol. Chem. 196: 669, 1952. 2. Drabkin, D. L., and Austin, J. H.: Spectrophotometric studies. II. Preparations from washed blood cells; nitric oxide hemoglobin and sulfhemoglobin, J. Biol. Chem. 112: 51, 1935. 3. Kruh, J., and Borsook, H.: Hemoglobin synthesis in rabbit reticulocytes in ~vitro, J. Biol. Chem. 220: 905, 1956. 4. London, I. M., Shemin, D., and Rittenberg, D.: Synthesis of heme in vitro by the immature non-nucleated mammalian erythrocyte, J. Biol. Chem. 183: 749, 1950. 5. Borsook, H., Deasy, C. L., Haagen-Smit, A. J., Keighley, G., and Lowy, P. H.: Incorporation in vitro of labeled amino acids into rat diaphragm proteins, J. Biol. Chem. 186: 309, 1950. 6. Dubnoff, J. W.: An apparatus for the incubation of tissue slices and homogenates, Arch. Biochem. 17: 327, 1948. 7. Krebs, H. A., and Henseleit, K.: Untersuchungen uber die Harnstofibildung im Tierkorper, Z. Physiol. Chem. 210: 33, 1932. 8. Roche, J., Derrien, Y., and Moutte, M.: Solubility in concentrated saline solutions and specific characteristics of blood hemoglobins, Bull. Soc. chim. biol., Trav. Mem. 23: 1114, 1941. 9. Schapira, G., Dreyfus, J. C., and Kroh, J.: Recherches sur la biochimie de l'hemo$10bine a ['aide du fer radioactif. 1. Fractionnement des hemoglobines de lapin adulte par denaturation alcaline, Bull. Soc. chim. biol. 33: 812, 1951. 10. Kruh, J., Dreyfus, J. C., and Schapira, G.: Recherches sur la biochimie de

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128 PART II. BIOSYNTHESIS OF HEMOGLOBIN 15. 17. l'hemoglobine a [' aide de fer ra dioactif. v. Biosynth es e de s hemoglobines in vitro, Bull. Soc. chim. biol. 35: 1181, 1953. 11. Anson, M. L., and Mirsky, A. E.: Protein coagulation and its reversal. The preparation of insoluble globin, soluble globin and heme, J. Gen. Physiol. 13: 469, 1930. 12. Schack, J., and Clark, W. M.: Metalloporphyrins VI. Cycles of changes in sys- tems containing heme, J. Biol. Chem. 171: 143, 1947. 13. a) Shemin, D., and Wittenberg, J.: The mechanism of porphyrin formation. The role of the tricarboxylic acid cycle, J. Biol. Chem. 192: 315, 1951. b ) Shemin, D., and Kumin, S.: The mechanism of porphyrin formation. The formation of a succinyl intermediate from succinate, J. Biol. Chem. 198: 827, 1952. 14. Schroeder, W. A., Kay, L. M., and Wells, I. C.: Amino acid composition of hemo- globins of normal negroes and sicl~le-cell anemics, J. Biol. Chem. 187: 221, 1950. Tristram, G. P.: The amino acid composition of proteins, in Neurath, H., and Bailey, K., The Proteins, 1: 180, Academic Press. 1953. 1 6. Cohen, P. P., and Hayano, M.: Urea synthesis by liver homogenates, J. Viol. Chem. 166: 251, 1946. Fontes, G., and Thivolle, L.: Le tryptophane et l'histidine vent des acides amines hematogenes, Compt. rend. Acad. 191: 1088, 1930; Recherches experimentales sur les processus chimiques de l'hematopo~ese et sur la pathogenic des enemies, Le Sang 4: 658, 1930. 1 8. Okugawa, Y., and Tatsui, M.: Uber die Wirkung des Tryptophans auf experi- mentelle Anamie und ihre Beziehung zur Milz, Z. Physiol. Chem. 195: 192, 1931. 19. Matsuoka, A., and Nakao, T.: Uber die Wirkung des Methyltryptophans auf kunstliche Anamie und Ernahrung, Z. Physiol. Chem. 195: 208, 1931. 20. a) Drabkin, D. L., and Miller, H. K.: Hemoglobin production, II. The relief of anemia due to milk diet, by feeding amino acids, J. Biol. Chem. 90: 531, 1931; b) Hemoglobin production III. The relief of anemia due to milk diet, by feed- ing amino acids and related compounds, J. Biol. Chem. 93: 39, 1931. 21. Elvehjem, C. A., Steenbock, H., and Hart, E. B.: Ineffectiveness of purified glu- tamic acid as a supplement to iron in the correction of nutritional anemia, J. Biol. Chem. 93: 197, 1931. 22. Keil, H. L., and Nelson, V. E.: The effect of oral administration of amino acids and intraperitoneal injection of various elements and hydrochloric acid on re- generation of hemoglobin, J. Biol. Chem. 97: 115, 1932. 23. Alcock, R. S.: The role of tryptophan in blood development, Biochem. J. 27: 754, 1933. 24. Kotake, Y.: Zum intermediaren Sto~wechsel des Tryptophans, Ergeb. Physiol. 37: 245, 1935. 25. Hamada, T.: Zur F`rage der hamatopoetischen Wirkung des Tryptophans, Z. Physiol. Chem. 243: 258, 1936. 26. Pearson, E>. B., Elvehj em, C. A., and Hart, E. B.: The relation of protein ~.o hemoglobin building, J. Biol. Chem. 119: 749, 1937. 27. Chin Kyu-sui: Uber den Einflusz des a-N-Methyltryptophans (Abrins) auf kunstliche Anamie und auf die Ernahrung, Z. Physiol. Chem. 257: 18, 1938-39. 28. Whipple, G. H., and Robscheit-Robbins, F. S.: a) Amino acids (natural and syn- thetic) as influencing hemoglobin production in anemia, Proc. Soc. Exp. Biol. and Med. 36: 629, 1937; b) Amino acids and hemoglobin production in anemia, J. Exp. Med. 71: 569, 1940.

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HEMOGLOBIN SYNTHESIS IN RETICULOCYTES BORSOOK 129 29. Harris, H. A., Neuberger, A., and Sanger, F.: Lysine deficiency in young rats, Biochem. J. :'7: 508, 1943. 3 0. Albanese, A. A., Holt, L. E., Jr., Kaj di, C. N., and Frankston, J. E.: Observa- tions on tryptophane deficiency in rats. Chemical and morphological changes in the blood, I. Biol. Chem. 148: 299, 1943. 31. Gillespie, M., Neuberger, A., and Webster, T. A.: Further studies on lysine de- ficiency in rats, Biochem. J. 39: 203, 1945. 32. Orten, A. IJ., and Orten, ). M.: A study of hemoglobin formation following the administration of certain amino acids to rats fed a diet low in protein, J. Nutr. 30: 137, 1945. 33. Kornberg, A.: Amino acids in the production of granulocytes in rats. Federation Proc. 5: 142, 1946. 34. Yeshoda, K. M., and Damodaran, M.: Amino acids and proteins in haemoglobin formation. I. Tryptophan, Biochem. J. 41: 382, 1947. 35. Daft, F. S.: Pteroylglutamic acid ("folio acid"), liver extract and amino acids in treatment of granulocytopenia in rats, Pub. Health Rep., U.S.P.H.S. 62: 1785, 1 947. 36. Orten, J. M., Bourque, J. E., and Orten, A. U.: The inability of human or beef globin to support normal hematopoiesis in the rat without added isoleucine, J. Biol. Chem. 160: 43 5, 1945. 37. Sebrell, H.: Anemias caused primarily by malnutrition, Federation Proc. 8: 568, 1949. 38. Nizet, A., and Robscheit-Robbins, F. S.: Reticulocyte ripening in experimental anemia and hypoproteinemia. Effect of amino acids in vitro, Blood, 5: 648, 1950. 39. Chandran, K., and Damodaran, M.: Amino acids and proteins in haemoglobin formation. 2. Isoleucine, Biochem. J. 49: 393, 1951. 40. Cannon, P. R., Steffee, C. H., Frazier, L. J., Rowley, D. A., and Stepto, P. C.: The influence of the time of ingestion of essential amino acids upon utilization in tissue-synthesis, Federation Proc. 6: 390, 1947. 41. Geiger, E.: Experiments with delayed supplementation of incomplete amino acid mixtures, J. Nutrit. 34: 97, 1947. 42. Geiger, E.: The role of the time factor in feeding supplementary proteins, J. Nutrit. j'6: 813, 1948. 43. Geiger, E.: The importance of the time element in feeding of growing rats: Experiments with delayed supplementation of protein, Science 108: 42, 1948. 44. Geiger, E.: The role of the time factor in protein synthesis, Science 111: 594, 1950. 45. Geiger, E.: Extra caloric function of dietary components in relation to protein ... . _ ~ . ~ , ~ _ A utilization, Federation Proc. 10: 670, 1951. 46. Geiger, E., Hagerty, E. B., and Gatchell, lI. D.: Transformation of tryptophan to nicotinic acid investigated with delayed supplementation of tryptophan. Arch. Biochem. 23: 315, 1949. 47. Harte, R. H., Travers, J. J., and Sarich, P.: The effect on rat growth of alternated protein intakes, J. Nutrit. 35: 287, 1948. 48. Henderson, R. and Harris, R. S.: Concurrent feeding of amino acids, Federation Proc. 8: 385, 1949. 49. Sanadi, D. R., and Greenberg, D. M.: Effect of amino acid deficiencies on in- corporation of radioactive carbon-labeled amino acids into animal tissue pro- teins, Proc. Soc. Exp. Biol. and Med. 69: 162, 1948. 50. Schaeder, A. J., and Geiger, E.: Cataract development in animals with delayed supplementation of tryptophane, Proc. Soc. Exp. Biol. and Med. 66: 309, 1947. 5 1. Tarver, H., and Schmidt, C. L. A.: Radioactive sulfur studies. I. Synthesis of