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OCR for page 111
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 RETICULOCYTES—BORSOOK 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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Representative terms from entire chapter:
amino acid
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
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.
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 10—c 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
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 10—5 1 0.2 X 10—4~ 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
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 ~; 10—I- 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.
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.
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-
HEMOGLOBIN SYNTHESIS IN RETICULOCYTES—BORSOOK 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
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
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.
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