new UNIFORMITIES :94 THE 8125 '
msmaunon as THE NASCENT CHAINS mom “5;? 3".-‘73ij;
FROM RABBIT Rmcumcms . g; 5259;, 3.;
Dissertafim for the Degree of Ph. B. ‘ ‘
macaw sma'a uwvvzasm _‘
ALBERTO PROTZEI.
' 1973‘." "
'fifi-‘Es...
ABSTRACT
NON UNIFORMITIES IN THE SIZE DISTRIBUTION
OF THE NASCENT CHAINS OF GLOBIN
FROM RABBIT RETICULOCYTES
By
Alberto Protzel
Evidence is presented to show that the nascent chains
of rabbit globin do not have a uniform distribution of sizes.
Data are presented to show that rabbit reticulocyte
ribosomes contain a significant component of completed a
globin which is still attached to tRNA (a globyl tRNA).
Additional data are presented to show that contamination by
labeled supernatant hemoglobin or labeled a globin from the
free globin pool present in reticulocytes is not a signifi-
cant factor in these results.
Some h.6% of the nascent a globin chains are present
as a globyl tRNA, instead of 0.71% as predicted on the basis
of the assumption that the size distribution of nascent
globin chains is uniform. On the other hand 8 globyl tRNA
comprises 0.69% of the nascent B globin chains. This value
coincides closely with the predicted value for nascent 8
globin chains uniformly distributed in size along the
polysome. Further evidence is presented to show that both
a globyl tRNA and 8 globyl tRNA exhibit the kinetic pro-
perties expected for normal intermediates of soluble
($.2551J5J Alberto Protzel
hemoglobin biosynthesis following inhibition of the initia-
tion of protein synthesis by pactamycin.
Radioactively labeled nascent chains of rabbit globin
were also analyzed as a function of molecular weight by gel
filtration on Bio Gel A-0.5M. The gel filtration analysis
showed peaks of radioactivity for peptides in the molecular
weight ranges of 10fl82-885H, 5891-6707, u573-u99u and 3068-
#088.
Gel filtration was done with Bio Gel A-0.5M, 10%
agarose, mesh ZOO-U00. The gel was equilibrated in 6M
guanidine HCl-0.l B mercaptoethanol. All elutions were
done with this same solvent at a pressure differential of
57-60 cm of solvent. Calibration was done with peptide
markers covering the molecular weight range from 356 to
16000.
The accumulation of nascent peptides of globin at
discrete ranges of molecular weight indicates that the
rate of movement of ribosomes along the mRNA of rabbit
globin is not uniform.
NON UNIFORMITIES IN THE SIZE DISTRIBUTION
OF THE NASCENT CHAINS OF GLOBIN
FROM RABBIT RETICULOCYTES
By
Alberto Protzel
A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Biochemistry
1973
ACKNOWLEDGEMENTS
The author wishes to thank Dr. Allan J. Morris for his
interest and guidance which have made this thesis possible.
Miss Maureen McCully furnished valuable technical assistance.
The author also thanks Dr. Willis A. Wood and Dr. Hyram Kitchen
for amino acid analyses. Thanks are due to my wife Chris for
patience and cooperation during the course of my research.
ii
TABLE OF
LIST OF TABLES . . . . .
LIST OF FIGURES . . . .
LIST OF ABBREVIATIONS .
INTRODUCTORY STATEMENT .
LITERATURE REVIEW . . .
CONTENTS
Translational Models of Control
The Reticulocyte .
0
Control of Hemoglobin Biosynthesis
Reticulocyte mRNA
Rates of Growth of
Chains
Control of Release
Chains
Role of Heme
Role of tRNA .
MATERIALS AND METHODS .
1.
2.
3.
A.
5.
6.
7.
8.
Reagents o o o o o
Pretreatment of Reticulocytes for
Labeling . .
Labeling of Reticulocytes
Preparation of Ribosomal Pellets
the a and
of a and 86
Preparation of Peptidyl tRNA . .
Preparation of Urea Stock
Solutions
Preparation of Urea Buffers
Demo...
Bio-Gel P-lO Column Chromatography
DEAR-Cellulose Chromatography
Preparation of Globin Uniformly Labeled
C] or [ 3H] Tyrosine
Separation of Uniformly Labeled Alpha-
and Beta-Globin Chains
Analysis of Nascent Globin Chains
a. Analysis of a and B Globyl-tRNA
Pretreatment of Peptidyl-tRNA
for Tryptic Digestion
with [
iii
oooSoHoooo
Page
vi
vii
\oxooocn ON H
11
12
15
l7
l9
19
20
21
23
23
26
26
29
29
30
31
32
32
32
RESULTS .
l.
2.
3.
Tryptic Digestion . . . . . .
Separation of Tryptic Peptides .
Counting of Radioactivity . . . .
Analysis of Nascent Globin Chains
by Gel Filtration . . . . .
Recrystallization of Guanidine
Hydrochloride . . . . . . . .
Bio-Gel A- 0. 5M Gel Filtration
Chromatography . . . . . .
Treatment of the Sample for Bio-Gel
A- 0. 5M Gel Filtration . . . . .
Cyanogen Bromide Cleaveage of
Globin Chains . . . . . . . . .
Removal of Guanidine and B-ME from
Peptides o o o o o o a
Treatment of Data from Bio-Gel A- 0.5M
Gel Filtration Chromatography .
PlOtting Of Data 0 o o o o c 0
Construction of Theoretical
Curves . . . . .
Smoothing of Bio-Gel Filtration
ElUtion Data 0 o o o o o
Purified Peptidyl-tRNA is Free of Con-
tamination with Soluble Hemoglobin .
Accumulation of the Completed a Chain on
the POlyribcsome o o o o o o o o o
Labeling of the Ribosomes in the
Wh01e RGtiCUloyte o o o o o o 0
Determination of the Amount of a
and B Globyl-tRNA . . . . . . .
EffeCtOfHeminoooooooo o.
Pactamycin Induced Decay of Radio-
activity in the Nascent
GIObin Chains o o o o o o o o o
Accumulation of Growing Globin Chains on
the Polyribosome . . . . . . . . .
Calibration of the 810- Gel A- 0. 5M
a.
Gel Filtration Column . . . . .
Peptide Markers . . . . . . . .
Calibration of the Column . . .
Identification of the Column
MarkeI‘S.........
Analysis of Peptides D, E,
F and G O I O O O O O 0
Significance of Peak X
in Figure 11 o o o o o 0
Analysis of Peptides H, I
andJoooooo
The Calibration Curve .
iv
35
“5
156
146
“7
118
A9
A9
50
SO
51
53
5H
51:
5a
5a
23
65
73
71:
7t:
75
75
79
86
89
b. Nonuni
DISCUSSION . . . .
Accumulation
Accumulation
on the
Significance
Distri
REFERENCES . . . .
APPENDIX I . . . .
formity in Size Distribution
in the Population of Nascent
GlObin Chains. o o o o o o
Nascent Chains Labeled with
TerSine oooooooo
Nascent Chains Labeled with
Tryptophan, full Medium .
Effect of RNAse on the Elution
Pattern of Nascent
ChainSooooooooo
Nascent Chains from Whole
BlOOdoooooooooo
Labeling with Methionine . . .
of the Completed 0 Chain . . .
of Growing Globin Chains
Polyribosome . . . . . . . . .
of Nonuniformity in the Size
bution of Nascent Peptides . .
89
89
105
108
108
113
125
125
129
133
138
1145
Table
I.
II.
III.
IV.
V.
VI.
VII.
LIST OF TABLES
Incubation of Reticulocytes According to
Lingrel and Borsook (1963). . . . . . . . .
Final Concentration of Amino Acids in the
Modified Reaction Mixture of Lingrel and
BorSOOk (1963). o o o o o o o o o o o o o o
Added [3H] Hemoglobin Found in the Purified
Peptidyl tRNA FraCtion o o o o o o o o o 0
Analysis of [3H] Tyrosine Labeled Tryptic
Peptides from Purified Peptidyl tRNA . . .
Amino Acid Analysis of Peptides D, E, F,
and G, from Figure 11 o o o o o o o o o o 0
Distribution Coefficients (K ) of the
Marker Peptides as Measured n Figures
11 and 12 o o o o o o o o o o o o o o o o 0
Molecular Weights of the Peaks in Figures
19’ 22’ 2“. 25, 27 and 28 o o o o o o o o 0
vi
Page
2“
25
56
6A
78
96
12A
LIST OF FIGURES
Figure Page
1. DEAR-cellulose step during preparation
of peptidyl tRNA , 28
2. Separation of a and 3 chains of rabbit
g10bin O O O O O O O O O O 0 O O O O O O O O O 3)"
3. Calibration of a Bio-Gel P-lO column for
removal of Trypsin from a tryptic digest
of radioactive globin 38
A. Removal of trypsin from a tryptic digest
or [3H] globin O O O O O O C O O O O I O 0 O 0 no
5. Separation of tyrosine-containing tryptic
peptides from rabbit globin by two-
dimensional high voltage electrophoresis
and paper chromatography , , , , . , , . . . , A3
6. Time course of incorporation of [3H]
tyrosine into soluble hemoglobin of
rabbit reticulocytes , , , , , , , , . , , . , 58
7. Relative Specific activities of the nascent
globin peptides from purified peptidyl
tRNA , , 61
8. Effect of pactamycin addition to reticulo-
cytes labeled in the steady state . . . . , , 67
9. Pactamycin induced decay of radioactivity
in the 6 tyrosine-containing tryptic
peptides of rabbit globin , , , . , . . , . . 7O
10. Ratios of radioactivity found in tryptic
peptides from the N-terminal and C-terminal
portions of the nascent protein fraction
following pactamycin addition , , , , . , . . 72
ll. Bio-Gel A-0.5M agarose gel filtration
analysis of peptides for calibration of
the column
0 O O O O O O O O O O O O O O O O O 77
vii
Figure
l2.
13.
1A.
15.
16.
17.
l8.
19.
20.
21.
22.
LIST OF FIGURES (cont.)
Bio-Gel A-0.5M agarose gel filtration
analysis of peptides for calibration of
the column in the presence of tRNA . . .
Bio-Gel A—0.5M gel filtration analysis
of the peptides obtained from rabbit
globin by cleaveage with cyanOgen bromide
under mild conditions . . . . . . . . .
Bio-Gel A-0.5M gel filtration analysis of
the peptides obtained from rabbit globin
by cleaveage with cyanogen bromide under
Strong conditions 0 O I O O O O O O O 0
Identification of pooled samples H and I
from Figure 11 . . . . . . . . . . . . .
Identification of pooled sample J from
Figure 11 . . . . . . . . . . . . . . .
Calibration of the Bio-Gel A-0.5M column
or gel filtration in the absence of
peptidyl-tRNA O O O O O O O O O O O O 0
Calibration of Bio-Gel A-0.5M column for
gel filtration in the presence of
peptidyl-tRNA o o o o o o o o o o o o o
Bio-Gel A-0.5M gel filtration analysis
of the [3H] tyrosine-labeled nascent
peptides of rabbit globin . . . . . . .
Theoretical elution pattern for a popu-
lation of [3H] tyrosine-labeled peptides
from globin analyzed by Bio-Gel A—0.5M
get filtration chromatography . . . . .
Bio-Gel A-0.5M gel filtration analysis
of the [3H] tyrosine-labeled peptides
of rabbit globin . . . . . . . . . . . .
Bio-Gel A-0.5M gel filtration analysis
of the [3H] tryptophan-labeled nascent
peptides of rabbit globin . . . . . . .
viii
Page
81
83
85
88
91
93
95
100
102
IOU
107
Figure
23.
2A.
25.
26.
27.
28.
29.
LIST OF FIGURES (cont.)
Theoretical elution pattern for a popu-
lation of [3H] tryptophan-labeled nascent
peptides from globin analyzed by Bio-Gel
A-0.5M gel filtration chromatography . .
Bio-Gel A-0.5M gel filtration analysis of
the [3H] tryptophan-labeled nascent pep-
tides of rabbit globin after treatment
with pancreatic RNAse . . . . . . . . . .
Bio-Gel A-0.5M gel filtration analysis of
the [3H] tryptophan-labeled nascent pep-
tides of rabbit globin synthesized in
Whale blOOd o o o o o o o o o o o o o o 0
Time course of incorporation of [3H]
tyrosine into rabbit reticulocytes in the
absence and in the presence of methionine
Bio-Gel A-0.5M gel filtration of [353]
labeled nascent peptides of rabbit globin
With Standard lEUCine o o o o o a o o o o
Bio-Gel A-0.5M gel filtration of [’53]
labeled nascent peptides of rabbit globin
With lmM LGUCine o o o o o o o o o o o o
Hypothetical population of 10 nascent
peptides uniformly distributed in size .
ix
Page
110
112
115
118
120
123
132
LIST OF ABBREVIATIONS
CM-cellulose carboxy methyl cellulose
DEAE-Cellulose diethylaminoethyl cellulose
B-ME 2-mercaptoethanol
mRNA messenger ribonucleic acid
poly A polyadenylic acid
poly T polythymidilic acid
POPOP l,A-bis 2-(A-methyl-5-phenyloxazolyl)
~benzene
PPO 2,5- diphenyloxazole
BDS sodium dodecyl sulphate
tRNA transfer ribonucleic acid
INTRODUCTORY STATEMENT
It has been shown by Dintzis (1961) that the assembly
of polypeptide chains of hemoglobin takes place by the se-
quential addition of amino acids, starting at the N-terminal
end and continuing towards the C-terminal end of the poly-
peptide chain. A physical basis for this assembly process
is provided by the polyribosome, a multiple ribosome struc-
ture (Warner‘gt.§l., 1963, Rich g£,§l., 1963).
Translation of genetic information into proteins
(Nirenberg and Matthaei, 1961), is accomplished in the
polyribosome (Rich gt_al., 1963), by stepwise (Erbe, Nau and
Leder, 1969) movement of the ribosome along the mRNA
(Lengyel £3 21., 1973) while carrying one nascent polypep-
tide chain (Warner and Rich, 196A).
Measurements of the rate of movement of the ribosome
along the mRNA have been made by various authors. Thus,
translation of the tryptophan operon of Escherichia 321;,
takes place at the rate of approximately 1000 nucleotides
per minute at 30° (Morseg£,§1., 1969). These authors
compared the kinetics of appearance of both mRNA and of
enzyme activity to obtain these results. The rates of
elongation of egg white proteins have been determined by
Palmuter (1972) by measuring the time required for
radioactivity first observed as nascent peptides to appear
as supernatant protein. This author estimates a translation
rate of 900 nucleotides per minute for ovalbumin at A1°. A
similar technique has been applied by Lodish and Jacobsen
(1972) to the measurement of the rate of elongation of the
a and B chains of hemoglobin. These authors observed a
translation rate of 131 nucleotides per minute at 25° for
both chains of hemoglobin. All these methods provide average
values for the rate of ribosomal movement leaving unanswered
the question about the relative rates of translation of
specific portions of the mRNA.
The problem of relative rates of movement along the
mRNA can be also studied by analyzing the size distribution
of nascent peptides in polyribosomes that have achieved a
steady state of synthesis. The distribution of sizes will
be uniform if ribosomes go past every codon at the same
rate. While if a ribosome spends a great deal of time at a
given codon the correSponding nascent peptides will be pre-
sent in an increased amount. There will be more poly-
ribosomes with a ribosome present at that particular codon.
Several authors have studied the size distribution of
nascent peptides of globin by means of Naughton - Dintzis
plots (Naughton and Dintzis, 1962). According to this
method, a population of nascent chains that is labeled with
a certain radioactive amino acid is prepared. The specific
activity of the amino acid at each one of its position
of occurrence is determined. The specific activity data
3
are then plotted against the number that corresponds to the
position along the chain at which the measurement was made.
A straight line plot indicates a uniform size distribution.
This method was applied by Hunt gt al. (1968a) and by Luppis
23.31. (1970) to populations of nascent chains from reti-
culocyte polysomes. These authors concluded that the nascent
chains of hemoglobin are uniformly distributed in size and
that therefore the rate of ribosome movement along the mRNA
of globin is constant. This thesis presents evidence that
there are deviations from a uniform distribution in size
for the nascent chains of hemoglobin. Hence, the rate of
ribosome movement along the mRNA is not uniform.
Those nascent chains still attached to tRNA are re-
ferred to in this thesis as peptidyl tRNA, while those
nascent peptides attached to tRNA whose primary amino acid
sequences are those of the completed globin chains are re-
ferred to as globyl tRNA.
A procedure for the purification of the peptidyl tRNA
component from rabbit reticulocyte ribosomes has been de-
scribed by Slabaugh and Morris (1970). This method is par-
ticularly effective in removing soluble hemoglobin con-
tamination from the peptidyl tRNA preparation.
The availability of this methodology has made feasible
studies of the size distribution of the nascent chains of
hemoglobin. A uniform distribution in size for the chains
obained from this peptidyl tRNA implies that ribosomes move
at a constant rate along the mRNA. As discussed above, this
A
thesis asks the question: Do the ribosomes move at a con-
stant rate along the mRNA for globin?
Plan of the theSis.
After confirming that the method of Slabaugh and
Morris (1970) does indeed give preparations of peptidyl-
tRNA essentially free of soluble hemoglobin, the fraction
corresponding to a globyl-tRNA and B globyl-tRNA was mea-
sured in that preparation.
The a and B globin chains of rabbit hemoglobin each
contain 3 tyrosine residues in their amino acid sequence
(Dayhoff and Eck, 1968). The C-terminal ends of a and B
globin molecules consist of the amino acid sequences -Lys
-Tyr-Arg and -Lys-Tyr-His respectively. Since the bio-
synthesis of hemoglobin is known to proceed from the N-
terminal end toward the C-terminal end (Dintzis, 1961) an
analysis of the purified peptidyl-tRNA fraction for the
presence of the C-terminal dipeptides tyrosyl-arginine and
tyrosyl-histidine, following tryptic digestion, has per-
mitted a determination of the amounts of a globyl-tRNA and
B globyl-tRNA in that fraction.
While B'globyl-tRNA exists to the extent predicted by
a uniform distribution of peptides the a globyl-tRNA was
found to be present in an amount 6 times greater than the
theoretical value predicted on the basis of the assumption
of a uniform distribution of sizes of nascent a globin
peptides. The size distribution of the remainder of the
5
population of nascent chains has been studied by means of a
column chromatographic procedure. A Bio-Gel A0.5M agarose
gel column has been calibrated for molecular weight deter-
mination of peptides ranging in molecular weight from 316
to 16000 daltons. This column procedure permits the dis-
play of the nascent peptides of hemoglobin as a function of
molecular weight, thus allowing one to single out particular
segments of the mRNA where ribosome movement might be slower
or faster than others.
LITERATURE REVIEW
Translational Models Of Control
Inherent to the differentiated state is the production
of cell specific proteins. The biosynthesis of these cell
specific proteins is usually associated with long lived
messages. The mRNA for cocoonase has a half life of 100
hours (Kafatos, 1972), ovalbumin, 18 hours (Palmiter 32
al., 1973). In the loach Misgurnus'fossilis information
issued by embryo nuclei in the middle blastula stage is
realized only in the course of gastrulation (Spirin, 1969).
Reticulocytes can synthesize hemoglobin for at least A8
hours after extrusion of the nucleus (Rifkind gt gl., 196A).
Various models have been presented to account for the
levels of proteins in eucaryote tissues. Kafatos (1972)
attributes a key role to differential mRNA stability to
account for specific protein levels that are high, in the
presence of little or no gene amplification. Schimke (1970),
emphasizes rates of degradation of enzymes. In more recent
work, Palmiter and Schimke (1973). present the concept that
more efficient translation of a long lived mRNA would lead
to an increased rate of production of its corresponding
polipeptide. This increase in efficiency would be due to
more favorable competition for some rate limiting factor
7
once the more labile messages start to disappear. Any fac-
tor that would decrease the level of short lived messages
would lead to superinduction of proteins with long lived
mRNA. Sussman (1970), presents a "ticketing" theory to
account for both qualitative and quantitative levels of
proteins given the existence of long lived messages. Quan-
titative control would be achieved by allowing the ribosome
to clip-off a particular "ticket codon" after a round of
translation. After a certain number of clippings the mes-
sage would be susceptible to RNAse attack. Qualitative
control would be achieved by having at a given time a
ribosome population capable of reading only certain mes-
sages. The ticketing theory might have some basis. Mea-
surements of the length of poly-adenylic acid, poly (A),
segments in mRNA of HeLa cells have been performed by
Sheiness and Darnell (1973). These authors labeled the
cells briefly with 3H-adenosine, and transferred them to
fresh nonradioactive medium. They observed a maximum
shortening from 200 to 100 nucleotides in the poly (A) of
the mRNA between 3 and 6 hours after transfer to fresh
medium. In long term experiments, they observed pieces
only 50 adenylate residues long in the poly (A). Similar
ticketing phenomena might be operating in the case of hemo-
globin mRNA. Duck erythrocytes labeled for A hours with
[’H] adenosine have poly (A) segments of at least 150
nucleotides in length (Pemberton and Baglioni, 1972).
Messenger RNA from rabbit reticulocytes labeled for 18
8
hours before removal from the animal had poly (A) sequences
50-70 nucleotides in length (Lim and Canellakis, 1970).
Furthermore mRNA from circulating reticulocytes, older than
18 hours, was found to have poly (A) sequences of 8 resi-
dues (Burr and Lingrel, 1971).
Tomkins gt 31. (1969) have presented a model based on
inducers and repressors to explain their results of the
steroid mediated induction of liver tyrosine amino trans-
ferase. They suggest that the sole role of the steroid is
to antagonize a post transcriptional repressor which both
inhibits messenger translation and promotes messenger de-
gradation. The reticulocyte presents a convenient system
for the study of models of translational control, as shall
be discussed below.
The Reticulocyte
Over 90% of the protein synthesized by the reticulo-
cyte is hemoglobin. In addition to hemoglobin, reticulo-
cytes synthesize six other proteins, two of which are mem-
brane proteins (Lodish, 1973 a, b). The degree of com-
partmentation of protein synthesis in the reticulocyte is
presumably very limited. Disk gel electrophoresis patterns
of proteins synthesized by whole reticulocytes and by mem-
brane free lysates are identical Lodish (1973a). These
observations reflect the progressive specialization of the
reticulocyte as it becomes an erythrocyte.
The reticulocyte is the last step of erythroid cell
9
differentiation that is morphologically distinct from the
erythrocyte (Ram, 1969). Erythropoiesis starts with the
multipotential stem cell, capable of becoming committed to
differentiation into erythrocytes, leucocytes or megacaryo-
cytes which give rise to the platelets (LaJtha, g£_al., 1971).
Further differentiation leads to a succession of morpholo-
gical states, designated according to their varying staining
capacities. These states are in succession, the proery-
throblast, baSOphilic erythroblast, polychromatophilic
erythroblast and the orthochromatic erythroblast. Extru-
sion of the nucleus by the orthochromatic erythroblast leads
to the reticulocyte (Ham, 1969; Tarbutt and Blackett, 1968).
The rate of synthesis of hemoglobin is maximum at the
polychromatOphilic stage of development. These results
were obtained by measuring authoradiographically the uptake
of S’Fe by cells from the hepatic erythroid population of
the mouse (DJaldetti 35 al., 1970). Reticulocytes are cells
in the process of degeneration. The number of ribosomes
in marrow erythroid cells is 5.A times that in reticulocytes
(Lingrel and Borsook, 1963).
Control of Hemoglobin Biosynthesis
Reticulocyte mRNA
The mRNA for globin has been widely sought for. Labrie
(1969) reported the finding of a 108 RNA species. Its
specific activity was five times higher than that of 188,
29s and 53 RNA 17 hours after injection of 32Po~ into the
10
rabbit. Its T1 RNAse digest did not correspond to that of
18S or 29S RNA. Lockard and Lingrel (1972) reported the
preparation of mouse 98 RNA capable of synthesizing the a
and B globin chains of the mouse in a duck reticulocyte
lysate system. Williamson 93.21. (1971) reported the pre-
paration of mRNA from mouse reticulocytes having a molecular
weight of 170,000, which would correspond to 65 nucleotides
in excess of the number necessary to code for a polypeptide
the size of globin. This mRNA from mouse has been trans-
lated in a mouse liver S-30 cell free system giving a and B
chains in a ratio of 1.5 to 1 (Sampson 32 91., 1972). Poly
(A) sequences will bind to millipore filters and to poly [T]
sequences. Brawerman gt_§1. (1972) have obtained prepara-
tions enriched in 108 RNA by passing crude preparations of
polysomal RNA through millipore filters. Similar prepara-
tions have been passed through oligothymydilic acid-cellulose
columns to obtain a 98 RNA capable of synthesizing rabbit
globin in a Krebs II ascites tumor cell free system (Aviv and
Leder, 1972). Gianni g£_§1. (1972) have prepared 10$ RNA
from the post ribosomal supernatant of rabbit reticulocytes
which will only direct synthesis of a globin chains in a 308
supernatant of rat liver. A similar protein synthesizing
system programmed with 108 RNA obtained from the polysomes
by these authors, yielded both a and B chains in a ratio of
1.5 to 1 respectively. Jacobs-Lorena and Baglioni (1972)
have isolated a 203 ribonucleOprotein from reticulocyte
post ribosomal supernatant that gives 108 RNA. A Krebs II
11
ascites cell-free system programmed with this mRNA gives
only a chains while the same system using reticulocyte
polysomal mRNA gives a ratio of a to B chains of 0.A8.
Similar results have been obtained by Housman 2£.§l- (1971)
using the same incubation system. The widely varying ef-
ficiency of heterologous cell-free systems for synthesizing
the a and 8 chains of hemoglobin can be compared to the
reticulocyte itself. Reticulocytes produce about equal
ratios of the a and B chains of hemoglobin (Baglioni and
Colombo, 196A). These results obtained with heterolOgous
systems programmed with reticulocyte mRNA might reflect the
effect of supernatant cofactors on the rate of mRNA trans-
lation.
Rates of growth of the a and B Globin Chains
The direction of elongation of proteins is from the N-
terminal end to the C-terminal end, Dintzis (1961). This
means that the last amino acid added to a protein before
release would be expected to be the C-terminal amino acid.
One would thus expect that shortly after the addition of
a radioactive amino acid to reticulocytes synthesizing
hemoglobin, the amino acids towards the carboxy end of the
supernatant hemoglobin would have more radioactivity (Dintzis,
1961).
The time lag between the first appearance of radio-
activity at the carboxy end and the first appearance of
radioactivity at the N-terminal end represents the time it
12
took to assemble and release the protein (Knopf and Lamfrom,
1965). In practice, the method of KnOpf and Lamfrom (1965)
measures the time it takes for tryptic peptides labeled with
the same amino acid and situated at opposite extremes of the
protein to reach the same specific activity. Using this
method, Hunt g£_al. (1969) have found that the a chain of
globin is translated on the average 70% faster than the 8
chain of globin.
Lodish and Jacobsen (1971) have criticized these re-
sults on the grounds that the label was not being incor-
porated at the same rate into all of the peptides studied.
If label were being inserted by a degenerate pair of tRNA's
(3011 gt a1., 1966) each charged with the same amino acid
but at different specific activities, mistakenly high or
low rates of translation would be obtained. High Specific
activity for the aminoacyl-tRNA at the amino end would pro-
duce a rate of elongation shorter than the actual rate and
vice versa. To avoid these artifacts Lodish and Jacobsen
(1971) concentrated on a given peptide and measured the time
lag between the first time it was observed to incorporate
label and the time it appeared as part of a completed solu-
ble protein. By using this approach an elongation time of
200 seconds per chain and a rate of release of 15 seconds
per chain was found for both chains, at 25°.
Control of Release of a and B Chains
The striking equality of the amounts of a and B chains
13
synthesized by the reticulocyte led to the suggestion that
there was an interrelationship between the synthesis of the
a and 6 chains. Balanced synthesis has been observed for
example for the A and B subunites of tryptOphan synthetase
in £1.9211 but these are produced by a polycistronic mes-
senger (Morse gt_al., 1969). In the case of the a and B
globin chains, the corresponding genes are not linked
(Itano, 1960). The molecular weight of the 103 RNA that
codes for globin is around 170,000 - 190,000 (Williams gt
21., 1971; Labrie, 1969). This range of molecular weights
corresponds to a size of an average of 520 nucleotides, which
would code for a protein of around 170 amino acid residues,
assuming three nucleotides per codon (Nirenberg g£_al., 1965).
The a and B chains of globin have 1A1 and 146 amino acid
residues, respectively. Thus, the mRNA for globin, as iso-
lated, cannot contain coding information for both globin
chains in the same polynucleotide backbone.
To account for the balanced synthesis of the two
chains of hemoglobin Colombo and Baglioni (1966) proposed
that completed a chains aided the release of B chains. Many
studies have presented evidence against this view.
In the genetic disease u—thalassemia, characterized
by decreased synthesis and the a chain of hemoglobin H(B~)
can be detected (Motulsky, 196A). Hemoglobin H accumulates
and appears as inclusion bodies in older erythrocytes, show-
ing that 8 chain synthesis can proceed in the absence of a
chain synthesis (Motulsky, 196A). By the same token, studies
1A
in patients with B thalassemia show that a-chains will ac-
cumulate in the absence of 8 chain synthesis (Fessas,
1966). Honiget El. (1969) have selectively blocked syn-
thesis of the human a chain from fetal hemoglobin leaving
the synthesis of a chain unaffected as compared to controls.
These authors used the O-methyl threonine analog of isoleu-
cine which prevents its incorporation in hemoglobin (Hori
and Rabinovitz, 1968). Since the a chain of human fetal
hemoglobin has isoleucine (Dayhoff and Eck, 1968), while
the a chain does not, it is possible to selectively in-
hibit the growth of one chain. Rabinovitz 32 a1. (1969)
have performed a similar inhibition experiment using rabbit
reticulocytes. They have used a heterozygous rabbit in
which one half of the B chains have no isoleucine. The a
chains contain the three isoleucines found in normal rab-
bits while the remainder of the B chains have the normal
presence of l isoleucine. When the synthesis of a chains
was retarded to 10% of controls the formation of the
isoleucine-less 8 chain was stimulated by at least 30%.
Ascribing this stimulation of the variant 8 chains to in-
creased availability of limiting factors these authors con-
clude that each globin subunit is synthesized independently
of the other one.
Seemingly contradictory results concerning the in-
dependence of the rates of synthesis of the chains of
globin have been obtained by Schaeffer et a1. (1969).
These authors added human B chains to a hemoglobin
l5
synthesizing cell free system from rabbit reticulocytes.
There was a A0-50% decrease in the amount of radioactivity
of the rabbit 8 chain component in the supernatant fraction
and an increase in the amount of completed or almost com-
pleted B chains in the ribosome fraction.
Role of Heme
As shown by Kruh and Borsook (1956), there is a
parallelism in the rates of synthesis of heme and globin.
Murine proerythroblastoid cells (T-3-Cl-2) transformed by
Friend Leukemia virus show detectable amounts of globin
mRNA 2 days after induction with dimethyl sulphoxide. Glo-
bin mRNA reaches a maximum value A days after induction with
dimethyl sulphoxide (Ross 33 al., 1972). At this time a
hemoglobin like color can be detected (Friend gg_§1., 1971).
The synthesis of globin is dependent on the presence of iron,
a precursor of heme Borsook (1958). Removal of iron by
chelation will lead to polysome disagregation and cessation
of synthesis of hemoglobin (Rabinovitz and Waxman, 1965).
Heme has been shown to be implicated in hemoglobin
biosynthesis both at the level of initiation of translation
(Zucker and Schulman, 1968; Adamson gt $1., 1968) and at the
level of the completed chains by regulating the level of the
pool of free a chains (Tavill g£_al., 1972).
A role of heme in initiation was suggested by the ob-
servation that addition of hemin to an unfractionated
reticulocyte lysate increased the rate of initiation of
16
globin chains (Zucker and Schulman, 1968) and helped pro-
longue the linear rate of synthesis (Howard 23 a1., 1968).
Hemin was found to prevent the formation of an inhibitor
that would form during incubation at 37°. Addition of an
aliquot from a lysate incubated without hemin was able to
inhibit protein synthesis in a fresh, unincubated lysate
(Maxwell and Rabinovitz, 1969; Howard EE.E£°: 1970). This
effect was shown to be temperature dependent (Hunt 23.31.,
1972). At temperatures over 28° the rate of hemoglobin
synthesis has declined markedly by 10 minutes in a lysate
incubated without hemin. At 23° the rate does not decline
until after 30 minutes. Gross and Rabinovitz (1972) have
presented evidence that this inhibitor might exist in two
states, as a reversible inhibitor and as an irreversible
inhibitor. The reversible inhibitor would be in equilibrium
with a proinhibitor that is stabilized by hemin. Absence
of hemin would displace the equilibrium to reversible in-
hibitor which then would transform into the irreversible
inhibitor. Legon gt a1. (1973) have shown with sucrose
gradients that incubation of a lysate with 35S methionine
and hemin produces a complex between met-tRNA and the A08
ribosomal subunit. A similar incubation without hemin showed
a rapid disappearance of the complex after two minutes of
incubation, coupled with cessation of protein biosynthesis.
This sparing effect of heme is not limited however to the
initiation of globin chains. Lodish and Desalu (1973) have
17
observed that reticulocyte lysates incubated in the absence
of hemin show a depression in the synthesis not only of
globin but in the synthesis of the six other major proteins
known to be produced in the reticulocyte and are unable to
synthesize any of the 8 known reovirus-specific proteins.
McDowell 22.210 (1972) have shown that the complete lysate
system will synthesize the 8 reovirus proteins. These re-
sults on the scOpe of the heme effect have been confirmed by
Mathews 22.91. (1973) using mRNA for mouse globin, calf lens
crystallins and the RNA from EMC virus. In all these cases
heme stimulates the synthesis of the corresponding proteins
in the rabbit reticulocyte lysate.
Effect of tRNA
Changing patterns of isoaccepting tRNA species or of
levels in a given species of tRNA have been associated with
changes in differentiation. Benzoylated DEAE cellulose
column chromatography has shown the presence of the tRNAlyS
isoaccepting species (tRNAllyS, tRNAzlyS) in vegetative or
sporulating Bacillus subtilis, while in spores tRNAzlys is
missing or found in very low concentrations (Chuang and D01,
1972). Methylated Albumin Kieselguhr (MAK) chromatographic
profiles of tRNAmet and tRNAarg in erythrocytes of larval
bull frog (Rana catesbeiana) differ from that found in the
adult erythrocytes (DeWitt, 1971).
In cells committed to synthesis of specific proteins,
the tRNA population tends to correlate with the amino acid
18
composition of the proteins being synthesized. Sheep reticu-
1ocytes contain different levels of tRNAile and tRNAmet in
accordance with the particular allele of the 8 chain of
hemoglobin that is being synthesized (Litt and Kabat, 1972).
Transfer RNA from rabbit reticulocytes contains a high ratio
of acceptance activity for histidine as compared to isoleu-
cine. Histidine is very frequent in hemoglobin and isoleu-
cine is very frequent in hemoglobin and isoleucine is very
infrequent (Smith and McNamara, 1972). These authors find,
however, that leucine acceptance activity is unusually low
relative to the number of leucine residues present in hemo-
globin. The role of modulator tRNA (Ames and Hartman, 1963)
in rabbit reticulocytes awaits further studies.
MATERIALS AND METHODS
1. Reagents
Cycloheximide, bovine hemin (2x crystallized) and
ribonuclease A (5x crystallized, protease free) from bovine
pancreas were purchased from Sigma Chemical Company, St.
Louis, Middouri. Sparsomycin was generously donated by
Drug Research and Development, Division of Cancer Treatment,
National Cancer Institute, Bethesda, Maryland. Trypsin
treated with L-(l-Tosylamido-2-phenyl) ethyl chloromethyl
ketone was obtained from Worthington Biochemical Corporation,
Freehold, New Jersey. Pactamycin was donated by the Upjohn
Company, Kalamazoo, Michigan. Penicillin G was purchased
from Nutritional Biochemicals Corporation, Cleveland, Ohio.
Streptomycin Sulfate, U.S.P., was acquired from General
Biochemicals, Chagrin Falls, Ohio. Diethylaminoethyl
cellulose (DE-52) and carboxymethyl cellulose (CM-32) were
purchased from H. Reeve Angel and Company, Clifton, New
Jersey, and Bio Gel P-lO was from Bio Rad Laboratories,
Richmond, Ca. Aquasol and Liquifluor were obtained from
New England Nuclear, Boston, L-["S] Methionine and L-[3,5
~3H] Tyrosine were purchased from Amersham/Searle Corpora-
tion, Arlington Heights, Illinois. Specific activities
ranged from 33 to A0 Ci per mole for tyrosine and from A0
19
20
to 133 Ci per mole for methionine, respectively. L-[aH]
Tryptophan (7.10) mole was purchased from Schwarz/Mann,
Orangeburg, New York. L-[170] Tyrosine, 455 C1 per mole,
was ordered from Schwarz/Mann, Orangeburg, New York. Nitro-
cellulose filters (0.A u pore size) were from Schleicher
and Schuell Company, Keene, New Hampshire. The synthetic
dipeptides, L-tyrosyl-L-arginine and L-tyrosyl-L-histidine,
were prepared by Cyclo Chemical, Los Angeles, California.
Bio Gel -A 0.5M, 200-A00 mesh, 10% agarose, was purchased
from Bio-Rad Laboratories, Richmond, California. Blue
Dextran 2000 was purchased from Pharmacia. DNP-alanine
was kindly furnished by Dr. R. J. Evans of Michigan State
University. Guanidine HCl was purchased from Sigma, grade
I. All other reagents used were reagent grade.
2. Pretreatment of Reticulocytes for Labeling
Male New Zealand rabbits were made reticulocytic by
four daily subcutaneous injections of 2.5% phenylhydrazine.
The rabbits received no injections on days 5 and 6. The
phenylhydrazine was dissolved in an isotonic solution con-
taining 0.13 M NaCl, 5.2 mM KCl and 7.5 mM MgClz (NKM)
(Allen and Schweet, 1962). Following the addition of
glutathione to a final concentration of 10'"3 M, the pH
was adjusted to about 7.3. The resulting solution was
filtered and frozen until used. On day 7 of the injection
sequence the animals were given a light ether anesthesia
followed by an injection of 100 mg of Nembutal and 2000 I.U.
21
of heparin via the marginal ear vein. Blood was obtained
by heart puncture and the collected blood cooled immediately
to A°. Hematocrits ranged from 12 to 16. All subsequent
steps were carried at 4°. The blood was passed through
glass wool and the cells were separated from the plasma by
centrifugation for 20 minutes at 4000 X g in a Sorvall re-
frigerated centrifuge. The plasma was decanted and the
volume measured. The packed cells were then washed with a
volume of the "reticulocte saline," RS, described by Lingrel
and Borsook (1963), equal to the plasma volume. The RS
contained 0.13 M NaCl, 5 mM KCl and 7." mM MgClz.6 H20.
The cells were resuspended in a small volume of RS, the
remainder of the RS was added, the suSpension stirred and
the cells recovered by centrifugation for 20 minutes at
A000 X g. The washing procedure was identically repeated
once more and the cells recovered by centrifugation as
before.
3. Labeling of Reticulogytes
A suspension of reticulocytes was incubated in a
modified medium of Lingrel and Borsook (1963), Table 1.
Plasma from the same rabbit was dialyzed 1 hour against
35 volumes of cold RS prior to use in the incubation
medium. The amino acid mixture of Lingrel and Borsook
(1963) was used, except that hydroxyproline was omitted
and L-asparagine and L-leucine were added to a final con-
centration of 0.51 mM and 2.58 mM in the incubation medium
22
(Hunt, 1968). Final concentrations of all the amino acids
in full medium appear in Table II. Modifications were
made as indicated in the text and in the legends to figures.
These modifications were made depending on the particular
radioactive amino acid used in the labeling experiment.
When radioactive tyrosine was used, it was absent from the
medium until the radioisotope was added. Nonradioactive
L-tyrosine was added to the isotopically labeled tyrosine
to give a final concentration of 0.021 mM in the incubation
medium. This concentration of tyrosine was used for all
incubation unless otherwise indicated. When [’58] methionine
was used as a label, nonradioactive methionine was omitted
entirely from the medium. L-E’SS] methionine was added un-
diluted to the reaction mixture in amounts of 1 or 2 Mci
and specific radioactivities averaging 150 Ci/mole. Parti-
cular values used are indicated for specific experiments
in the legends to the figures. When tryptophan was used no
amino acids were omitted from the incubation mixture. All
incubations were performed at 37°. After an initial 2
minute warm-up period the radioactive amino acid was added
to the reaction mixture. This addition of radioactivity
defined zero time of incubation. The incubation was ter-
minated by pouring the entire incubation mixture or suitable
aliquots thereof into a 12 fold volume of ice cold RS con-
taining cycloheximide at a concentration of 16.5ug per m1,
(0.059 mM). The cells were then collected by centrifugation
and washed once with fresh RS containing cycloheximide.
23
A. PrgparatiOn of Ribosomal Pellets
The washed reticulocytes were lysed for 10 minutes
with A volumes of 2.5 mM MgCl2 containing 0.09 mM cyclo-
heximide and 0.21 mM Sparsomycin. The solution was then
made isotonic by the addition of one volume of 1.5 M
sucrose-0.15 M KCl. Cell debris was removed by centrifu-
gation at 2000 X g for 20 minutes. The supernatant solu-
tion was then centrifuged at 6A000 X g for 3 1/2 hours to
obtain the radioactive ribosomal pellets (1X). Where in-
dicated the ribosomal pellets were resuspended in medium
B (Allen and Scweet, 1962), and reisolated by sedimentation
as before to yield washed (2X) ribosomes. Medium B con-
tains 0.25 M sucrose, 17.5 mM KHCO, and 2 mM MgCl,. The
concentration of ribonucleoprotein was determined by mea-
suring the absorbance at 260 nm using an absorption co-
efficient of 11.3 for a concentration of 1 mg per m1 (Ts'O
2E.Elx: 1961).
5. Preparation of Peptidyl-tRNA
Ribosomal pellets were resuspended in a small volume
(approximately 1 m1) of 0.25 M sucrose containing 0.059 mM
cycloheximide and 0.1A mM Sparsomycin. The ribosomal sus-
pension was then used to prepare peptidyl-tRNA according to
the method of Slabaugh and Morris (1970). It was found
that reduction of the urea concentration of buffers I and
II from 8.0 to 7.6 avoids the occasional problem of crystal-
lization of the urea solutions atlfifi. This modification
Table I.
II.
21:
Incubation of Reticulocytes according to Lingrel
and Borsook (1963).
Reagent Mixture
In order of addition:
Component
Amino acid Mix.* In RS. ph 7.75
MgCl2 (0.25 M) plus Glucose 10%
TRIS.HC1 (0.16AM) pH 7.75
Sodium Citrate (10'3M) in plasma
Sodium Bicarbonate (10'3M) in plasma
Reaction Mixture
In order of addition:
Component
Reticulocytes (Packed cell volume)
Reagent Mixture
KFe(NH,)2(SO,)2.6HzO (10.5 mg/lOml)
in RS
Radioactive amino acid in RS
5A.00
2.70
27.00
21.60
32.A0
137.70
10.00
26.A0
A0.30
*Table II gives the concentrations of the amino acids
in the reaction mixture (final concentration).
Amino
acids are 3.893 times as concentrated in the stock
solution referred to as Amino Acid Mix.
25
Table II. Final Concentration of Amino Acids in the Modified
Reaction Mixture of Lingrel and Borsook (1963)*
*Any deviations from these final concentrations are indicated
in the legends to the figures or in Methods.
Amino Acid Concentration (mM)
Alanine 0.51A
Arginine 0.128
Asparagine 0.51A
Aspartic acid 0.732
Glycine 1.361
Histidine 0.617
Isoleucine 0.077
Leucine 2.569
Lysine 0.A62
Methionine 0.077
Phenylalanine 0.All
Proline 0.360
Serine 0.A2A
Threonine 0.A37
Tryptophan 0.077
Tyrosine 0.206
Valine 0.822
Cysteine 0.103
Glutamine 2.055
26
together with the utilization of 360 ml of Buffer 1 during
the "Buffer I wash" during DEAE-cellulose chromatography
have been employed through out this thesis, Figure 1. The
pooled fraction containing the purified peptidyl-tRNA was
reduced to a volume of approximately 1.8 ml by ultrafil-
tration in an Amicon cell with a UM-2 Diaflo membrane. The
concentrated sample was then dialyzed against 3 - 1500 ml
portions of deionized water, lyophilized and stored at -21°.
Prgparation of Urea Stock Solutions
Urea solutions (8.5A M) were prepared at room tempera-
ture and deionized by stirring with Amberlite MB-3 for
approximately 1 1/2 hours. The ion exchange resin was
removed by filtration and the resulting solution used as
a stock urea solution for the preparation of the other urea
containing solutions. A solution containing 6M LiCl and
7.6 M urea was prepared from this stock solution. Solid
LiCl was added slowly to an 8.5A M stock urea kept in an
ice bath. This solution was then kept at A° until used.
Prgparation of Urea Buffers
Buffer I contained 7.6 M urea, 0.1 M sodium acetate
pH 5.6 and 0.05 M 2-mercaptoethanol (2-ME). Buffer I
was prepared from stock solutions of 8.5A M urea, 5 M 2-ME
and glacial acetic acid. Buffer I was titrated to pH 5.6
at room temperature using 6N NaOH. Buffer II was identical
except it contained 0.75 M sodium acetate and was titrated
with saturated NaOH.
Figure l.
27
DEAE-cellulose step during preparation of pepti-
dyl-tRNA. A sample of [3H] tyrosine-labeled
ribosomes was treated with LiCl/urea and the
soluble fraction desalted on Bio-Gel P-lO as
described in the text. The desalted material
(35 ml, 10.76xlo6 dpm) from the Bio-Gel P10
column was applied to a DEAR-cellulose column,
as described in Methods. The sample was washed
with 350 ml of buffer 1. Buffer II was then
applied. Aliquots of each fraction were analyzed
as described in Methods.
28
l5
IO
3H CPM x IO'3
b... _-_
5 IO
'Buf'fer‘r éL-fiuffér 11'
_gg___ l
sos IO|520
FRACTION NUMBER
Figure 1
29
Bio-Gel'Palo Column Chromatography
Bio-Gel P-10, 50-100 Mesh, column chromatography was
used to remove LiCl from the solution containing peptidyl-
tRNA. Bio-Gel P—10, 9.5g was soaked overnight in approxi-
mately 250 m1 of Buffer I. The slurry was allowed to
settle for 15 minutes and the supernatant was removed by
aspiration. The total remaining P-lO was poured into a
column 1.9 cm in diameter to a bed height of 33 cm. Prior
to use the column was washed with 50 m1 of buffer I and run
at a flow rate of about 0.37 ml/min. Three m1 fractions
were collected.
DEAE-Cellulose Chromatogrgphy (Figure l)
Whatman De-52 microgranular cellulose was used. Seven
grams of the cellulose exchanger was suspended in 60 m1 of
0.5 N acetic acid and aspirated with agitation to remove
CO The slurry was titrated to pH 5.6 using 6 N NaOH, and
2.
the "fines" removed by allowing the cellulose to settle for
a number of minutes equal to 2.5 times the height of the
slurry in cm. The "fines" were then removed by aspiration
of the supernatant solution. Approximately 60 ml of buffer
I was added and the removal of fines repeated. This was
followed by two subsequent removals of fines with buffer 1.
The total remaining DEAE-cellulose was poured into a 2 cm
diameter column to give a bed height of approximately 6 cm.
Prior to use the column was washed with about 50 ml of buf-
fer I. The sample was applied to the column and the absorbed
30
peptidyl-tRNA was washed with at least 360 m1 of buffer I
at a flow rate of about 12 ml per 30 min.
6. Preparation of Globin Uniformly Labeled with
‘1'CJ'or {‘HJ'Tyrosine
Washed reticulocytes were incubated in the presence of
tyrosine labeled with the appropriate isotOpe as described
above. The tyrosine concentration in the medium was 0.1 mM.
Penicillin and streptomycin were added to the reaction mix-
ture to a final concentration on 0.11 mg per m1 of each.
Incubations were allowed to proceed at 37° for 3 1/2 to A
hours. The cells were washed, lysed and the post ribosomal
supernatant used to prepare hemoglobin according to the
method of Winterhalter and Huehns (196A). The post riboso-
mal supernatant was dialyzed at A° against 2-1 liter por-
tions of 0.01 M sodium phOSphate buffer, pH 6.8. The
dialyzed solution was applied to a CM-Sephadex column (1.8
x 20 cm), equilibrated with 0.01 m sodium phosphate pH 6.8.
This was followed by a wash with 100 ml of the same buffer.
Elution was done with a convex gradient formed by placing
250 m1 sodium phosphate pH 6.8 in a constant volume chamber
and adding 0.02 M Na,HPO,to the chamber while stirring.
Fractions containing hemoglobin were combined and dialyzed
for A8 hours against 3 portions (1 1t each) of deionized
water at A°. Hemoglobin was determined by the method of
Austin and Drabkin (1936). Globin was prepared by the cold
acid acetone method of Rossi Fanelli £2 a1. (1958). The
dialyzed hemoglobin (15 mg per ml) was added dropwise and
31
with magnetic stirring to 30 volumes of cold acetone con-
taining 6 mM HCL. The acetone HCL solution was kept cold
in dry ice - acetone bath, -86°. The precipitated globin
was collected by centrifugation at 1020 X g for 15 minutes
at ~20°. The supernatant was decanted and the globin dis-
solved in the minimal volume of deionized water. The globin
was dialyzed against 3-1 liter portions of deionized water
at A°. Recovery of radioactivity is approximately 79%. The
globin was stored at -20° as a lyophilized powder.
7. Separation of Uniformly_Labeled Alpha- and Beta-Globin
‘Chéins
The a and B globin chains of [1‘0] labeled rabbit
globin were separated on carboxymethyl cellulose (CM-32)
columns (1 x 22 cm) with a nonlinear gradient modified from
the procedure of Rabinovitz et al. (196A). The gradient was
generated by placing concentration multiples of 1,3,5,7,1,7
and 9 fold of the starting buffer (0.2 M formic acid - 0.02
M pyridine) in successive chambers of a 10 chamber rectangular
Varigrad (Buchler Instruments Inc., Fort Lee, New Jersey).
The contents of each chamber (50 ml) were 0.05 M in B-
mercaptoethanol (Lodish, 1971). Prior to chromatographic
separation the globin samples were dialyzed overnight
against 0.05 M B-mercaptoethanol and then adjusted to 0.2 M
formic acid, 0.02 M pyridine. Globin (A5 mg or less) was
applied to the column and eluted at a flow rate of lA-l6
ml per hour. The separated a and B globin chains were then
lyophilized and each was rechromatographed on a CM-32 column
32
by the same procedure in order to obtain further purification,
Figure 2. Lyophilized samples of separated globin chains were
stored at -20°. The purity of the separated a and B globin
chains obtained in this manner was established by the addi-
tion of nonradioactive carrier globin and digestion of the
mixture with trypsin at 37° for A hours as described below.
The six tyrosine containing peptides (aTA, aT6, cT15, BTA,
BTlA, BT16) were separated according to the method of Hunt
g£_§1. (1969) and analyzed for radioactivity. By this means
it could be shown that the a chain preparation contained
approximately 0.82% 8 chain while the 8 chain preparation
contained approximately 1% contamination by a globin.
8. Analysis of Nascent Globin Chains
a. Analysis of a and B Globyl-tRNA
Pretreatment of Peptidy1:tRNA for Tryptic Digestion
The lyophilized sample of labeled peptidyl-tRNA was
resuspended in 1.0 m1 of water containing 0.1 mg of pancreatic
RNase incubated at 37° for 25 minutes and 1y0philized. After
redissolving in 0.15 ml of 0.1 N NaOH the material was in-
cubated for 3 1/2 hours at 37° in order to cleave the
peptidyl-tRNA ester bond. The solution was then neutralized
with l N HCL to a pH of 5.A-5.6 as determined with pH indi-
cator paper. Purified a and 8 rabbit globin chains of known
radioactivity content (uniformly labeled with [1“CJ-tyrosine)
were then added as an internal standard. Nonradioactive
globin was added, if necessary, to give a mass of 3-A mg of
33
Figure 2. Separation of the a and 8 chains of rabbit globin.
A) Rabbit globin (A5 mg, 7.5 x 10“ cpm per mg) labeled uni-
formly with [‘“C] tyrosine was analyzed by CM-cellulose
chromatography as described in Methods.
B) Rabbit globin a chain obtained from A plus a chain ob-
tained from a similar experiment were pooled and re-
chromatographed as in A.
C) Same as in B except that rabbit globin 8 chain was used.
DO
"‘c CPM x lo"3
so 40 so so 70 so 90 IOO
FRACTION NUMBER
Figure 2
35
protein in the sample. The synthetic dipeptides, L-tyrosyl-
L-arginine (aTlS) and L-tyrosyl-L-histidine (BT16), were
added as carrier peptides (50 nmoles each) prior to tryptic
digestion.
Tryptic Digestion
Tryptic digestion was carried out in 0.1% NaHCO,,
Schapira et a1. (1968), at a final globin concentration of
3-A mg per m1. Trypsin was added in an amount equal to 2%
(w/w) of the total globin present. After 2 hours incubation
at 37°, 1% (w/w) trypsin was again added and the incubation
continued for an additional 2 hours. Samples were then
frozen and lyophilized. To prepare tyrosine labeled tryptic
peptides for agarose gel filtration chromatography, tryptic
digestion was done in 0.2 M ammonium bicarbonate (ABC) at a
final globin concentration of 1.3 mg per m1. Trypsin was
added in an amount equal to 1.3% (w/w) of the total purified
8 chain (16 mg) from rabbit globin uniformly labeled with
1"C -tyrosine. Incubation was continued for an additional
8 hours and the sample lyophilized.
Removal of trypsin seemed necessary to prevent any
possible hydrolysis of the larger peptide markers during
the pretreatment of the sample for gel filtration as
described further ahead. Besides it seemed desirable to
remove any large products of partial digestion which might
interfere with the identification of the larger peptide
markers. The tryptic digest was therefore purified by
36
passing through a Bio-Gel P-10 column. Results of one such
removal of trypsin are shown in Figure 3. Trypsin was as-
sumed to elute with the void volume of the column. Figure
A shows a similar experiment in which the tryptic peptide
markers for the experiment shown in Figure 11 were purified.
Separation of Tryptic Peptides
Separation of tyrosine containing tryptic peptides from
rabbit globin was performed by the two dimensional method of
Hunt gt El. (1969). which combines high voltage electro-
phoresis and paper chromatography, Figure 5. Tryptic peptides
are numbered according to their position of occurrence rela-
tive to the N-terminal end of the a and B globin chains of
rabbit hemoglobin (Gerald and Ingram, 1961).
The lyophilized sample containing the tryptic peptides
was dissolved in 0.1 m1 of 10% formic acid v/v and was ap-
plied to a 6 x 22 inch sheet of Whatman 3-MM Chromatography
paper in two 50 ul aliquots. The sample was applied as a
1 inch long streak along a line 5 cm away from the anode (-)
edge of the paper. The paper was wetted with the pH A.7
electrOphoresis buffer (Kitchen g§.§1., 1968), (1.25%
pyridine, 1.25% acetic acid) before electrOphoresis for
3.25 hours at 2000 volts. After drying the paper, a 5.0 cm
wide lane was cut containing the sample and scanned for peaks
of radioactivity by means of a Packard Radiochromatogram
paper strip scanner. The three areas of radioactivity were
detected. These areas are referred to as areas 1, 2 and 3,
Figure 3.
37
Calibration of Bio-Gel P-lO column for removal of
trypsin from a tryptic digest of radioactive glo-
bin. Rabbit globin (10 mg) uniformly labeled with
[3H] tyrosine was hydrolyzed in 0.2 M ammonium
bicarbonate with trypsin as described in methods.
The lyophilized tryptic digest was dissolved in
1 ml of 0.2 M ammonium bicarbonate. To this
solution 0.2 ml of blue dextran (18 mg/ml in 0.2
M ABC) and 1 drop of 0.1% (w/w) phenol red in
water was added. The sample was then applied to
a Bio-Gel P-lO column for analysis. Bio-Gel
P—lO was soaked overnight in 0.2 M ammonium
bicarbonate (0.2 M ABC). The Bio-Gel P-lO was
then poured into a 1 cm diameter column to give
a bed height of 27 cms. Three milliliter frac-
tions were collected and assayed for radioacti-
vity and for absorbance at 630 nm (blue dextran)
and 5A0 nm (phenol red). Aliquots, 0.5 ml, were
drawn from the odd numbered fractions and counted
in 5 ml of Bray's solution, see Methods.
38
131588.111eeonmdozqmmommq
o 8. . 6 4. 2
m I." O O. O O
_
_ — _ _ q
l5 ”
FRACTION NUMBER
Figure 3
_ _
2. 8 4
clone. x 28 I...
Figure A.
39
Removal of trypsin from a tryptic digest of [3H]-
globin. The B-chain (16 mg) of rabbit globin uni-
formly labeled with [3H] tyrosine was hydrolyzed
in 0.2 M ammonium bicarbonate containing 2% (w/w)
trypsin as described in Methods. This sample was
processed as in Figure 8 except that the blue dex-
tran and phenol red were omitted and the bed height
of the P-10 column was A0 cms. Odd numbered frac-
tions were assayed for radioactivity by diluting
aliquots (50 ul) with 0.5 m1 of water and counting
in Bray's solution. Fractions were pooled as in-
dicated by the horizontal line.
4O
mmmSSZ 20:04.1“.
0.
: onsmam
Om Om 0.? CM ON
a _
_ _ _
9
0| x WdO H
z...
Figure 5.
Al
Separation of tyrosine-containing tryptic peptides
from rabbit globin by two-dimensional high voltage
electrophoresis and paper chromatography. Ex-
perimental procedures appear in Methods. Non-
radioactive globin and the B-chain of rabbit glo-
bin uniformly labeled with [‘“C] tyrosine
(A0,000 cpm) were mixed and subjected to trypsin
digestion as described in Methods. Tyrosine
containing peptides appear cross hatched.
A. Strip scanner recording of the radioactivity
present in 8 following high voltage electro-
phoresis at pH A.7.
B. Paper strip containing tryptic peptides from
rabbit globin. The position of the origin and
the direction of movement of the tryptic peptides
during electrophoresis at pH A.7 are indicated
by an 0 and an arrow, respectively, between
panels A and B. The vertical wavy lines indicate
where segments containing radioactivity were cut
for further analysis in a second direction. From
left to right these segments are referred as areas
1, 2 and 3 as indicated by the numbers underneath.
C. Separation of the peptides in Area 1 by electro-
phoresis at pH 2 in the direction shown by the
A2
vertical arrow. The broken horizontal lines in-
dicate where the segment containing area was
sewed for separation in a second dimension. All
spots were first visualized with ninhydrin, see
Methods. Cross hatched spots were visualized by
further staining with a tyrosine Specific stain.
D. Separation of the peptides in area 2 by
descending paper chromatography. All other details
same as in panel C.
E. Separation of the peptides in area 3 by
electrophoresis at pH 8.9. All other details
same as in panel D.
43
CPM
00>
0 o 0
016'?) 0(6 0
0&1
“gag fl4@0 fl'ew
Figure 5
AA
from anode to cathode. The boundaries were marked and cut
to give 2 x A in rectangles. These rectangles were sewn to
A x 22 inch sheets of Whatman 3 MM chromatography paper.
The papers were then trimmed to preserve the overall length
of 22 inches. The sheet containing area 1 tryptic peptides
was wetted with pH 2 electrophoresis buffer (8% acetic acid,
2% formic acid) and run in the same buffer for 1 hour at
3000 volts. The sheet containing area 2 tryptic peptides
was placed in a cylindrical chromatography jar and the paper
equilibrated for 2 to 3 hours with the solvent system of
Waley and Watson (1953), (90:60:18:72::n-butanol:pyridine:
acetic acid:water, v/v). The chromatogram was developed
with this same solvent for 15 hours. The sheet containing
area 3 tryptic peptides was wetted with pH 8.9 electro-
phoresis buffer, (1% ammonium carbonate) and run in the
same buffer for 1.75 hours at 2000 volts. Peptides were
visualized by dipping through buffered ninhydrin containing
0.3% (w/v) ninhydrin in acetone which was 1% (v/v) in both
glacial acetic acid and pyridine (Easley, 1965). Tyrosine-
containing spots in parallel control runs were identified
using a l-nitroso-Z-naphtol stain. The paper was dipped in
a 0.1% (w/V) solution of this compound in acetone, dried
and dipped through acetone containing 10 ml concentrated
HNO3 per 100 ml. The appropriate areas containing the
radioactive tyrosine peptides were removed from the sheets,
remaining solvents removed in vacuo and the paper was then
A5
cut into small sectors and placed in scintillation vials for
the elution procedure. Three extractions with 2 ml of 0.01
N HCl were performed at 80°. The eluates were pooled into
fresh scintillation vials and lyophilized for counting of
radioactivity.
Countipg of Radioactivity
The tryptic peptides were dissolved in 0.01 N HCl and
combined with 10 ml of Aquasol and counted in a Packard liquid
scintillation spectrometer model 3310. Counting efficiencies
were determined by the channels ratio method for doubly
labeled samples. Counting efficiencies of samples contain-
ing a single-radioisotope were sometimes established by in-
ternal standardization with [’H] or [1‘0] labeled toluene
of known radioactivity content (New England Nuclear, Boston,
Mass.). Data expressed as decompositions per minute (DPM)
were determined from the observed cpm and the counting effi-
ciency.
The elution of radioactive materials during column
chromatography was monitored by placing 25-50 m1 aliquots
of the eluate fractions in 0.5 ml of H20, 5 m1 of Aquasol
and counting in a Nuclear Chicago, Unilux, liquid scintil-
lation counter.
Counting procedures for Agarose gel chromatography
are explained in the legends to the figures. Bray's solu-
tion (Bray, 1960) contained per liter: 60g naphtalene, Ag
PPO, 200 mg POPOP, 100 ml absolute methanol, 20 ml ethylene
A6
glycol and p-dioxane to volume.
b.‘ AnalysiS'of Nascent Globin Chains byGel Filtration
Reorystallization'Of Guanidine Hydrochloride
Guanidine hydrochloride was recrystallized according
to the method of Nozaki and Tanford (1967). Guanidine
hydrocholride, 500 g was dissolved with stirring in 2 liters
of absolute ethanol, kept close to its boiling point. Norite,
2 g, was added to the solution and stirred for 2 minutes.
Norite was removed by gravity filtration of the warm solu-
tion through two sheets of fluted filter paper. To the
clear solution 1.1 liters of benzene was added to precipi-
tate the guanidine hydrochloride. The precipitate was al-
lowed to stand overnight at A°. The crystals were harvested
in a Buchner funnel. The harvested crystals were trans-
ferred to a container which was placed in a vacuum desicator.
Benzene was removed by connecting the dessicator to a water
aspirator pump. The dry crystals were ground in a mortar
and placed under high vacuum. The dry guanidine hydro-
chloride crystals were further recrystallized from absolute
methanol. To 67 ml of absolute methanol near its boiling
point 100 g of guanidine hydrochloride was slowly added
with stirring. Any guanidine hydrochloride remaining un-
dissolved was brought into solution by adding small aliquots
of methanol. The warm methanolic solution was allowed to
stand overnight at A°. The crystals were harvested in a
Buchner funnel. A further crop of crystals was obtained by
A7
cooling the mother liquor in a dry-ice acetone bath. Excess
methanol was removed from the damp crystals by placing them
in a vacuum dessicator connected to a water aspirator pump.
The dry crystals were ground in a mortar and evaporated
under high vacuum in a lyophilizer to remove the last traces
of organic solvents.
Bio-Gel A-0.5M Gel Filtration Chromatogrgphy
Bio-Gel A-0.5M, (lots 10A75A and 11607), mesh 200-A00,
with a nominal agarose content of 10% was suspended in water
and allowed to settle several times to remove TRIS and
NaN3 added as a preservative. Water was decanted each time.
The agarose gel was then equilibrated with the eluting
solvent (6M guanidine HC1-0.lM B-ME, pH 6.5). ApprOpriate
amounts of dry guanidine HCl and B-ME were added to the
settled agarose gel. Following gentle swirling, enough
water was added to dissolve the guanidine HCl. The slurry
was degassed and litrated to pH 6.5 with 0.1N NaOH. The
agarose suSpension was then allowed to equilibrate over-
night at A°. All other Operations were performed at A°.
Gel beds ranging in height from 78 to 83 cm were formed
in Pharmacia K 15/90 columns. The columns were packed and
run under a pressure differential of 57-60 cm of solvent.
The pressure differential was maintained during the runs
with the use of a 500 ml Marriot flask. Flow rate was 3.0
ml per hour (1.73 ml per hour per cm”). Prior to use the
column was allowed to flow for at least 36 hours. To apply
A8
the sample, the column flow was momentarily stepped and the
sample, 0.2 ml, was layered under the solvent onto the tOp
of the gel with a Sage pump. Column flow was started im-
mediately after application of the sample. Fractions were
collected with a Gilson fraction collector. The fraction
size ranged from 11 drops (0.3 ml) to 26 drops depending
on the experiment, as indicated in the legends to the
figures. In other experiments 25 dr0ps were collected
with an 1800 Golden Retriever fraction collector. Frac-
tions were either collected into test tubes and aliquots
removed for counting Of radioactivity or directly into
scintillation vials and counted. In the latter case the
scintillation vials were fastened to the rotary table of
an 1300 fraction collector. In this latter case 11 drOps
were collected per fraction (0.3 m1). Positions of elution
of the blue dextran and DNP alanine markers were detected
by measuring their absorbance at 630 nm and 360 nm respec-
tively (Fish EE.§l'a 1969).
Treatment of the Sample for Bio-Gel A-0.5M Gel Filtration
A lyophilized preparation of peptidyl-tRNA was dis-
solved in 0.5 m1 of 0.1 N NaOH and was incubated for 3 1/2
hours at 37° in order to cleve the peptidyl-tRNA ester bond.
The solution was then neutralized with 1N HCl to a pH of
8.0-8.A as determined with pH indicator paper. The sample
was then 1y0philized. In experiments where peptidyl-tRNA
was not used this step was omitted.
A9
A 1y0philized sample containing free peptides was dis-
solved in 0.5 ml of column solvent, the pH was raised to 8.6
by adding 1N NaOH and the sample was allowed to stand at
room temperature for 2A hours to reduce disulphide bonds.
The pH of the solution was then lowered to pH 6.5 as de-
termined with pH indicator paper. At this time 70 ul of
3.6% blue dextran in column solvent was added followed by
80 ul of 0.1% DNP-alanine in 60% sucrose prepared in column
solvent. The clear solution was then centrifuged at 12000
x g to remove dust particles and 0.21 ml were loaded onto the
Bio-Gel A-0.5 m column.
CyanOgen Bromide Cleaveage of Globin Chains
Globin or separated globin chains were dissolved in
70% formic acid (v/v) at a concentration of 5 mg per m1
(Schroeder 22.21., 1968). Depending on the experiment, CNBr
was present in amounts ranging from 90 to 500 moles of CNBr
per mole of methionine present. Reaction times ranged from
18 hours to 72 hours. Reactions were carried in the dark.
At the end of the reaction, the reaction mixture was diluted
with 10 times its volume of water and 1y0philized.
Removal of Guanidine and B-ME from Peptides
The guanidine HCL - 0.1 M B-ME solvent used for Bio-
Gel A-0.5M chromatography was removed by chromatography in
columns packed with Bio-Gel P-2. Bio-Gel P-2 was swolen
overnight in 0.2M Ammonium Bicarbonate. Gel beds 1.2 x A0
cms were formed in glass columns. The fractions to be
50
analyzed were applied directly to the top of the gel bed and
eluted with 0.2 M ammonium bicarbonate. Three m1 fractions
were collected. Peptides were detected by counting of radio-
activity and guanidine H01 by measurement of conductivity
in a Radiometer conductivity apparatus. The fractions
containing the radioactive peptides were pooled and
lyophilized.
Treatment of Data from Bio-Gel A-0.5 M Gel Filtration
Chromatography
Plotting of Data
Elution data from Bio-Gel A—O.5 gel filtration
chromatography was presented according to Fish g£.§1. (1969).
Elution volumes corresponding to different experiments were
normalized by using the distribution coefficient (Kd), cor-
responding to each fraction as a representation of the posi-
tion of elution. The distribution coefficient (Kd) as
defined by Fish 22 31, (1969) is
Ve" V0
51" io
Kd ’
Where Xe is the weight of solvent used to elute a given
compound.
Xi is the weight of solvent contained within and with-
out the gel.
!0 is the void volume, i.e. the weight of solvent in
the column, external to the gel matrix.
Blue dextran 2000 was used to determine the exclusion
volume, by monitoring its absorbance at 630 nm. In this
51
thesis the fraction number containing the maximum absorbance
at 630 nm is defined as the void volume. Vi was determined
by monitoring the absorbance of DNP-alanine at 360 nm. The
fraction with an absorbance maximum at 360 nm was defined as
11' ‘1.
The cube root of the distribution coefficient, (Kd)‘/3,
V is any fraction between V and V
-e -o
of known peptide markers have been plotted against their
molecular weight raised to the 0.555 power (Fish 32 g1.,
1969). Straight lines have been obtained from these plots.
The slopes and intercepts of these straight lines have been
calculated by the least squares method.
Using distribution coefficients determined in two
separate agarose gel filtration analyses (Table VI) and
the molecular weights of the peptides as determined by
their amino acid sequence (Dayhoff and Eck, 1968) the
following linear relationship has been obtained:
Kd1/3 = 1.0568 - 0.002101 x M°'555 [1]
This relationship has been used to relate molecular weight
to distribution coefficient for each fraction.
Construction of Theoretical Curves
To interpret the observed elution pattern of nascent
globin chains, hypothetical elution curves have been computed.
These curves are based on the following assumptions.
a. The distribution of sizes of nascent peptides is uni-
form. This assumption defines a base line to compare
52
the experimental values.
b.
C.
The ratio of nascent a chains to nascent 6 chains is 1.
Hunt g£.§;. (1968a,b) find an average ratio of about
1.1. This thesis find a ratio of 1.0A.
The relationship between the distribution coefficient of
each nascent peptide and its molecular weight is given
by [l], as determined by calibration of the column.
The elution pattern of any single peptide will be a
Gaussian curve. This gaussian curve will be centered
at the Kd value of its corresponding peptide. The con-
tribution from each peptide is independent of the con-
tribution of the other peptides.
For a given labeled amino acid, the area under the
elution pattern of a peptide carrying this amino acid
will be proportional to the number of residues of this
amino acid present in the peptide.
Nascent a chain peptides and 8 chain peptides with the
same number of residues will have the same distribution
coefficient. This is a simplifying assumption used to
permit calculation of the Kd values in steps.
Molecular weight increase in steps of 110 the average
weight per residue for the 8 chain of hemoglobin. Es-
sentially the method used is equivalent to drawing a
gaussian curve with a maximum at the Kd value corres-
ponding to each nascent chain and adding the ordinates
of the resulting curves for each value of Kd. This
gives the composite elution pattern as a function of
53
Kd. A computer program implementing this idea is shown
in Appendix I.
Smoothing of Bio-Gel Filtration Elution Data
In one instance, figure 21, excessive fluctuation of
elution data was removed by the data smoothing procedure of
Savitzky and Golay (196A). A point was chosen along the
elution pattern. Four successive points were taken imme-
diately to the right and to the left of the chosen point.
A quadratic polynomial was then fitted by least squares to
the nine points. From the known abscissa of the chosen point
and the computed quadratic a new ordinate was computed for
the chosen point. The coefficients for a nine point fitting
listed by Savitzky and Golay (196A) were used. This fitting
procedure was repeated for all points of the elution pattern
except for the last four at either extreme of the elution
pattern.
RESULTS
1. ‘Purified Peptidyl-tRNA is Free of Contamination with
Soluble Hemoglobin
The analyses conducted in this thesis require that
purified peptidyl-tRNA be free of significant amounts of
contamination by soluble (labeled) hemoglobin. The two
analyses described below were performed to assess this
degree of contamination.
A mixture of nonradioactive reticulocyte ribosomes
and purified [3H] labeled hemoglobin was prepared (See
Legend of Table 3). This mixture was then subjected to
the procedure for preparation of peptidyl-tRNA, Slabaugh
and Morris (1970). Radioactivity present in the purified
peptidyl-tRNA fraction thus represents the extent of con-
tamination by hemoglobin in that fraction. Results from
the two separate analyses appear in Table 3. These re-
sults indicate that not more than 0.030% of the labeled
hemoglobin originally added remains in the purified
peptidyl-tRNA fraction.
2. Accumulation of the Completed a Chain on the Polyribosome
Labeling of the Ribosomes in the Whole Reticuloyte
Figure 6 shows the time course of incorporation of
[3H] tyrosine into the ribosomes and into soluble hemoglobin
5A
55
Legend
(Table III)
Rabbit reticulocytes (0.5 ml packed cell volume) were
incubated as described in Methods. The tyrosine concentra-
tion in the incubation medium was 0.1 mM. Labeled alanine,
valine and leucine (0.5 m Ci each) were added and the incu-
bation was allowed to proceed for A5 minutes at 37°. The
post ribosomal supernatant was dialyzed against 0.1 M
sodium acetate (pH 5.6) and passed through a DEAE-cellulose
column (1.5 x 5 cm) which had been equilibrated with the
same buffer. The labeled hemoglobin was then further
purified by CM-cellulose chromatography (see Methods).
Unlabeled 2x ribosomes in 0.25 M sucrose (21.A mg/ml) were
then combined with the purified [3H] hemoglobin (7.A x 106
DPM/mg) and purified peptidyl-tRNA was prepared. Samples
were counted by liquid scintillation using Bray's solution.
56
Table III Added 3H Hemoglobin Found in the Purified Peptidyl-
tRNA Fraction
‘Experiment [3H] Hemoglobin Unlabeled [3H] Hemoglobin re-
added ribosomes covered in the puri-
added fied peptidyl-tRNA
fraction
DPM x 10" mg DPM %
I 25.8 A0.0 7,850 0.030
II 18.1 A8.A u,u5o 0.025
Figure 6.
57
Time course of incorporation of [3H] tyrosine
into soluble hemoglobin of rabbit reticulocytes.
Rabbit reticulocytes (5 m1 packed cell volume)
were incubated as described in Methods. At
zero time 0.1 m Ci of 3H tyrosine (2A0 u Ci
per mole) was added to a final concentration of
0.021 mM. At the time points indicated 3 ml
aliquots were withdrawn from the reaction mix-
ture. The specific activity of the soluble
hemoglobin and of the ribosomes was measured in
the post ribosomal supernatant fraction and in the
twice washed (2X) ribosomes obtained from each
aliquot. Solutions containing 0.5 mg of hemo-
globin in 1 m1 of water or 0.090 mg of ribo-
nucleoprotein in 1 m1 of 0.25 M sucrose were
precipitated with an equal volume of 20% trichloro-
acetic acid. The precipitates were collected on
nitrocellulose membranes and counted in a toluene
Liquifluor mixture.
58
o—oIbw/WdO)Z_OIx NIBiOBd EINIOVOIOVB 3190103
MINUTES
to Q’ N
I I I I I I I do
.- F0 N
- o D -9
- o 49
" C o «In
‘ I.
I l 1 l 1 I
-V 70 N —
o—oIfiw/wemgpl x Ail/\llDVOIOVB ONnos ‘IVINOSOBIH
. FIFurc 6
59
of intact reticulocytes. The incorporation of radioactivity
into ribosomes reached a constant value by A minutes after
addition of [’H] tyrosine to the incubation medium. The
specific radioactivity of the ribosomes remained constant
for at least the next 16 minutes. The incorporation of
radioactivity into soluble hemoglobin was linear for at
least the first 20 minutes of incorporation. The constant
level of radioactivity found in the ribosomal fraction after
A minutes of incubation assures that a steady state of
labeling of precursor pools and nascent protein has taken
place. Nascent globin chains prepared from cells collected
at 10 minutes of incubation thus possess uniform specific
activity of the 6 tyrosine residues present in the nascent
globin chains.
Determination of the Amount of a and B Globyl-tRNA
Rabbit reticulocytes were incubated in a medium con-
taining E’H] tyrosine for 10 minutes at 37°. The ribosomal
pellets obtained from the labeled reticulocytes were used
to prepare the purified peptidyl-tRNA fraction. Following
the addition of [‘”01 labeled a and B globin chains to the
peptidyl-tRNA as internal standards, the mixture was digested
with trypsin and the tyrosine containing tryptic peptides
were isolated and analyzed as described in Methods.
The relative Specific activities ([3HJ/[1”C] ratio) of
the tryptic peptides are shown in Figure 7. The [’HJ/[1“C]
intercepts were calculated by the method of least squares
Figure 7.
60
Relative specific activities of the nascent globin
peptides from purified peptidyl-tRNA. The ordi-
nate represents the [3HJ/[‘“C] ratios obtained
in experiment I of Table IV. Each tryptic pep-
tide is positioned on the abscissa according to
the position of the C-terminal amino acid of
that tyrosine-containing tryptic peptide in the
sequence of rabbit hemoglobin. Tryptic peptides
have been numbered according to their position
of occurrence relative to the N-terminal end of
the corresponding rabbit globin chains. Lines
drawn through each set of points thus represent
the relative specific activities to be expected
for each amino acid present in a uniform distri-
bution of nascent chains on the polysome. The
[3H]/[1“C] intercept has been used as a measure
of the total nascent chains present and the ordi-
nate value corresponding to a T15 or B T16 has
been used as a measure of a globyl-tRNA or B
globyl-tRNA present, respectively.
61
,al4,aI6
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9 l0
(mp/(Hg) AllAllOV OHIOBdS BAIlV'IBH
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POSITION OF TRYPTIC PEPTIDE
VI ptur'o '/
62
in order to obtain a relative measurement of the number of
a and B nascent chains present, see Discussion. The [’H]/
[‘“C] ratios observed in the C-terminal tryptic peptides
(a T15 and B T16) were used as a relative measurement of
the number of completed a and B globin chains in the peptidyl-
tRNA fractions since only the completed globin chains in the
population of nascent chains can yield those tryptic pep—
tides upon hydrolysis, Dintzis (1961).
The results of three independent experiments are shown
in Table IV. Each set of experimental data was analyzed as
shown in Figure 7 for experiment I. It is apparent from
these data that rabbit reticulocyte ribosomes contain a
significant component of completed a globin which is still
attached to tRNA (a globyl-tRNA). Some A.6% of nascent d
globin chains are present as a globyl-tRNA. On the other
hand, completed B chains attached to tRNA (8 globyl-tRNA)
constitute only 0.70% of the nascent B globin chains.
Effect of Hemin
The presence of a pool of free soluble globin chains
has been shown to be present in the reticulocyte (Tavill
gg_§l., 1972; Baglioni and Campana, 1967). It has also
been reported that this pool is decreased in size if the
reticulocytes are incubated with hemin (Tavill gg’gl., 1972).
In order to examine the possible effects of hemin on the
accumulation of a globyl-tRNA on the ribosomes two paralled
incubations of rabbit reticulocytes were performed. One
63
LEGEND
(Table IV)
Rabbit reticulocytes (10 m1 packed cell volume) were
incubated for 10 minutes at 37°. The reaction mixture con-
tained 2 m Ci of [3H] tyrosine (2A21 u Ci/u mole). The in-
cubation conditions, preparation of peptidyl-tRNA, trypsin
digestion and analysis of labeled tryptic peptides are
described in detail in Methods. For each of the analyses
A7,A00 DPM of [‘“c] tyrosine labeled u-globin and 50,100
DPM of [‘”C] tyrosine labeled B-globin were added as a uni-
formly labeled internal standard.
6A
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97
components of such a population of globin chains according
to size, peptidyl-tRNA labeled with [3H] tyrosine was pre-
pared as described in Methods. The nascent globin chains
were released from the tRNA by treatment with 0.1 N NaOH
and then subjected to agarose gel filtration. Figure 19
presents the results of such an experiment. The eluted
radioactivity was plotted as a function of the distribution
coefficient corresponding to each eluted fraction. A
pattern of peaks is visible at Kd values of 0.28, 0.36, and
0.AA. Troughs are visible at Kd values of 0.32 and 0.A.
To assess the significance of the experimental curve a
theoretical elution curve for a population of nascent pep-
tides uniformly distributed in size was plotted, figure 20.
The construction of this curve has been discussed in the
Methods section. Points of insertion of tyrosine during
chain growth are indicated by arrows. Comparison of figures
19 and 20 shows that the overall range of Kd values dis-
played by both experimental and theoretical curves is very
similar, as expected. These values range from approximately
0.2 to about 0.7. The theoretical curve has a peak at Kd
0.25, corresponding to the peak at Kd of 0.28 of the experi- I
mental curve. No troughs or peaks are observed in the
theoretical curve. These results suggest that some members
of the population of nascent chains displayed in figure 19
are either decreased or increased in amount relative to the
other members of the population. Figure 21 shows a repeat
analysis of the same sample shown in figure 19. The same
Figure 19.
98
Bio-Gel A-0.5M gel filtration analysis of the
[3H] tyrosine-labeled nascent peptides of rabbit
globin. Rabbit reticulocytes (10 ml packed cell
volume) were incubated as described in Methods.
The incubation conditions were identical as those
described for labeling with [’H] tyrosine, except
that final concentration of leucine in the medium
was 1.0mM. At zero time 2 m Ci of [3H] tyrosine
(2A20 uCi per u mole) was added. After 10 minutes
of incubation incorporation of radioactivity was
stOpped as indicated in Methods and peptidyl-tRNA
was then prepared. An aliquot of [3H] tyrosine
labeled peptidyl-tRNA was then analyzed by Bio-Gel
A-0.5M gel filtration as described in Methods. The
effluent from column chromatography was collected
directly into scintillation vials fastened to an
ISCO rotary fraction collector. The rotary frac-
tion collector was actuated by a Gilson drOp
counter. Ten drop (approximately 0.3 ml) frac-
tions were collected. Radioactivity was assayed
by adding 0.2 ml of water to each scintillation
vial and counting in 5 ml of Aquasol. Counting
' was done in a Packard liquid scintillation
spectrometer model 3310. Counting efficiencies
were determined by the channels ratio method.
All data are presented as decompositions per
99
minute (DPM) as determined from the observed
cpm and the counting efficiency. To normalize
the data from different gel filtration analyses
the fraction number has been expressed as the
distribution coefficient (Kd) as described in
Methods. By definition, Kd for blue dextran is
O and Kd for DNP-alanine is 1.0.
100
l
*0
03
30-
25-
C)
01
K)
I
9.
,0I x waa H.E
I.0
0.5 06 0.7 0.8 0.9
0.4
0.2
0.I
Figure 19
0
III... .31. L
Figure 20.
101
Theoretical elution pattern for a population of
[3H] tyrosine-labeled nascent peptides from
globin analyzed by Bio-Gel A-0.5 gel filtration
chromatography. A population of nascent globin
chains with a uniform distribution in size has
been assumed. It has also been assumed that
there are equal numbers of nascent a-chains and
nascent B-chains. See Methods for further as-
sumptions. The theoretical curve has been
plotted as a function of Kd to facilitate com-
parison with the experimental curves Figures 19
and 21. See Methods for explanation of arbi-
trary units. The arrows indicate the position
of insertion of tyrosine residues along the
nascent chains.
102
_.O
1
I m3 0.
2
on. w. 03 K
om ohswfim
— F b h b _ b _ b _
SilNfl AHVBLIBBV
. llllllllllll I.)
111.1! 11':
Figure 21.
103
Bio-Gel A-0.5M gel filtration analysis of the
[3H] tyrosine labeled peptides of rabbit globin.
An aliquot of the sample analyzed in Figure 20
was analyzed identically. A Jagged curve was ob-
tained. This was smoothed out by a least squares
procedure (Savitzky and Golay, 196A) that filters
out noise. This procedure is described in Methods.
The smoothed data are presented as in figure 19.
104
I I I
I
07
l
0.6
I I
0.4 0.5
I
0.3
I
0.2
0.9
0.8
0.I
“3 G) Q'
g,0I x INdCI H,
Figure 21
105
pattern of troughs at Kd values of 0.32 and 0.A5 is observed.
Comparison of these two figures is important in connection
with the sharp peak of radioactivity present at a Kd value
close to 0. This peak would contain peptides with molecular
weight greater than 30,000. The patterns shown in figures
19 and 21 have the same general features in spite of the
very different sizes of the leading peak.
Nascent Chains Labeled with Tryptgphan, full Medium
Rabbit globin has two tryptOphan residues at positions
1A and 15 of the a chain reSpectively, and one residue at
position 37 of the B chain,(Dayhoff and Eck, 1968). These
amino acid residues inserted early during chain growth, make
tryptophan a convenient label for displaying a population of
nascent peptides. Tryptophan is no longer inserted past
residue 37 in the 8 chain. Thus, steep curves that might
obscure some peaks are avoided. Peptidyl-tRNA was therefore
prepared with reticulocytes incubated with a full complement .
of amino acids plus tritiated tryptOphan. Figure 22 shows 3
the population of nascent globin chains obtained in this I
experiment. Troughs at Kd values of 0.33 and 0.A5 are again
‘inj' in. ‘L ”n 1":-
observed, together with peaks of radioactivity at Kd values
of 0.29 and 0.37 plus a shoulder at Kd 0.62. Due to instru-
ment failure between Kd values of 0.A7 and 0.56 a dotted
line has been drawn suggesting a peak around 0.55. This
value was suggested by the SIOpes of the two limbs of the
incomplete peak. A theoretical curve for tryptophan labeling
Figure 22.
106
Bio—Gel A-0.5M gel filtration analysis of the
[3H] tryptOphan labeled nascent peptides of
rabbit globin. Rabbit reticulocytes (10 ml
packed cell volume) were incubated as indicated
in Methods for labeling with tryptophan. At
zero time 2 mCi of [3H] tryptophan (7100 uCi
per umole) was added. Following this addition
the specific activity of the A0.3 ml reaction
mixture was 2275 uCi per umole. After 10 minutes
of incubation, incorporation of radioactivity
was stopped as indicated in Methods and peptidyl-
tRNA was then prepared. An aliquot of [3H]
tryptophan-labeled peptidyl-tRNA was then ana-
lyzed by Bio-Gel A-0.5M gel filtration. Collecf
tion and analysis of fractions identical as in
Figure 19. Fractions between Kd values of 0.A7
and 0.56 were lost. These are indicated by
broken lines.
107
[93“; M541 HEREFEHHNIVIQ—
mm musmwm
. _ _ _
3
IN
3
In
_
_
1+»
_ 1 A.
I.
In
um
_ p p _
2, Ix wao H2
108
appears in figure 23. Again, as in the case of the theore-
tical curve for tyrosine, there are no troughs in the
theoretical curve for tryptOphan.
' Effect of RNAse on the Elution Pattern of Nascent Chains
A peak of radioactivity was observed to elute close
to the void volume in all Agarose gel filtration experiments.
Figures 19 and 21 have shown that the pattern of troughs
and peaks is independent of the size of this peak. The
possibility that this peak might represent RNA-polypeptide
complexes (Huang and Baltimore, 1970), was investigated.
An aliquot of the same sample of [3H] tryptophan peptidyl-
tRNA that appears in Figure 22 was incubated for 25 minutes
at 37° with protease-free pancreatic RNAse. After RNAse
treatment this sample was analyzed by Bio-Gel A-0.5M gel
filtration chromatography. The results are shown in Figure
2A. This pattern is nearly identical to that of Figure 22.
I" 3 h
Nascent Chains from Whole Blood
Rabbit reticulocytes kept at 0° for one hour show an
almost complete disappearance of polysomes. Upon warming
to 37° the polysome pattern is almost completely restored
f’T‘” .
after one minute and completely restored after two minutes
(Tepper and Wierenga, 1972). These same authors find an
oscillatory rate of hemoglobin synthesis in the precooled
reticulocytes. In reticulocytes kept at 37° prior to in-
cubation the rate of protein synthesis is linear. This
phenomenon was observed only with precooled reticulocytes.
A..-«I.E...h..nu~. u
Figure 23.
109
Theoretical elution pattern for a population of
[3H] tryptophan labeled nascent peptides from
globin analyzed by Bio-Gel A-0.5M gel filtra-
tion as described in Methods. The assumptions
and presentation of data are identical as in
Figure 20.
Dulat ion of
ides from
. filtra-
sumptions.
183 in
fll5
0c I4
I\
3"
Cl) I I
N E? 9
8.].an AHVHIIBBV
' 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 I .0
OJ
5
X
(n
(‘0'
C
E
E
L
‘AWnn '_ 1' L! ' .'_-:)I.'I._li) .l] .55 . k” “42: rs
Figure 2A.
111
Bio-Gel A-0.5M gel filtration analysis of the
[3H] tryptophan-labeled nascent peptides of
rabbit globin after treatment with pancreatic
RNAse. An aliquot of the sample shown in
Figure 23 was incubated with 0.1 mg of pan-
creatic RNAse (protease free), for 25 minutes
at 37° and analyzed as by Bio-Gel A-0.5M as in
figure 19.
112
l I
[0 N
€_Ol x wao H2
LG
02 03 0.4 0.5 0.6 0.7 0.8 09
0|
Figure 24
113
As reported by these authors a low degree of reticulocytosis
(30%) and attainment of final temperature within A0 seconds
are necessary to observe oscillations. In the cell incuba-
tions reported in this thesis, reticulocytes were kept under
A° for at least one hour and were brought up to 37° within
two minutes, at the most. The possibility existed that the
agarose gel filtration patterns were artifacts induced by
cold synchronization. To explore this possibility peptidyl-
tRNA was prepared from reticulocytes that had not been ex-
posed to low temperatures prior to incubation. Blood (A0
ml) was drawn from an anemic rabbit by heart puncture, fil-
tered and while still warm added to an Erlenmeyer flask con-
taining [3H] tryptOphan (3mc, 7.1 mCi/umole) dissolved in
0.5 ml of RS. The cells were incubated for 12 minutes at
37°. Peptidyl-tRNA was prepared from this incubation mix-
ture and analyzed by Bio-Gel A-0.5M gel filtration. The
results of this experiment appear in figure 25. The pattern
is essentially identical to that seen in figures 22 and 2A.
Therefore any results observed cannot be ascribed to syn-
chronization of ribosomes following exposure to the cold.
Labeling with Methionine
To study patterns of nascent peptides, methionine has
the advantage that it is inserted into globin during initia-
tion of protein synthesis (Wilson and Dintzis, 1969; Hunter
and Jackson, 1971; Koffer - Gutmann and Arnstein, 1973).
N-terminal methionine is removed during elongation of both
III I. III] 1). I'll
ill .ll llllll. III 1],)
f1).ll|llll
Figure 25 0
11A
Bio-Gel A-0.5M gel filtration analysis of the
[3H] tryptophan-labeled nascent peptides of
rabbit globin synthesized in whole blood.
Details of the experiment are discussed in
the text. Bio-Gel filtration analysis as in
figure 19.
115
0;
an.
may
say
_
may
may
mm ousmam
N.
0.
ON
z,0I x wao H2
116
the a and the B chains of rabbit globin. The stage of pro—
tein synthesis at which the removal of N—terminal methionine
takes place is not certain. Jackson and Hunter (1970) have
estimated that peptides between 15 and 20 amino acids long
loose their N-terminal methionine. Similar results were
obtained by Yoshida‘g§,§1. (1970) who found that peptides
shorter than 16 amino acids had N-terminal methionine.
Koffer-Gutman and Arnstein (1973) found N-terminal methionine
in peptides up to 50 amino acids long. There is one
methionine at residue 32 in the a chain and one at position
55 of the 6 chain of rabbit globin. Thus, assay for the
presence of nonuniformity in size distribution in the popu-
lation of nascent chains of globin can be done for all size
ranges if radioactive methionine is used as a label.
The attainment of steady state labeling of the cells
was verified, as shown in figure 26. As shown in this
figure a steady state of labeling had been achieved after
about eight minutes. The sample incubated without methionine
had achieved a steady state that was lower by 7%, than the
sample without methionine. Borsook (1957) has shown that
methionine is not rate limiting. Eleven minutes seems to
be an adequate point for collecting the cells. Figure 27
shows the effect of incubating reticulocytes in the pre-
sence of all amino acids except methionine. This figure
shows the same peaks that were observed with the [3H]
tyrosine and [3H] tryptOphan labeled peptides. Peaks at
Kd values of 0.289, 0.3Al, 0.A70 and 0.602 are observed.
Figure 26.
117
Time course of incorporation of [3H] tyrosine
into rabbit reticulocytes in the absence and
in the presence of methionine. Rabbit reticulo-
cytes (10 ml packed cell volume) were suspended
in the incubation medium described in Methods,
except that leucine was added to a final con-
centration of 1 mM in the incubation medium and
methionine and tyrosine were omitted. The cell
suspension was then divided in two aliquots.
At zero time the following additions were made.
To one aliquot [3H] tyrosine (30 HO per umole)
and methionine was added to give final concen-
trations of 0.1 mM and 0.077 mM respectively.
To the other aliquot only tyrosine was added to
the same Specific activity and concentration in
the final reaction mixture. At the time points
indicated A ml aliquots were withdrawn from the
reaction mixture. The Specific activity of the
ribosomes was measured in the twice washed (2X)
ribosomes obtained from each aliquot. Solutions
containing 0.21—0.25 mg of ribonucleoprotein in
1 m1 of 0.25 sucrose were precipitated with an
equal volume of 20% tricholoacetic acid. The
precipitates were collected on nitrocellulose
membranes and counted in a Toluene Liquifluor
mixture.
118
°—°(13W -)‘,_0I x (Em/was)
<1“ I!) N —
I I I I
20
o I°
REALXM,XKD,XKDI,SUM(1500),BUFF(1500),
YSTAR(200),YGAUSS(300)
INTEGER N,K,I.J,NCEL,NOCEL,INCK,IOLFAC,
IFAC,IFLAG
INITIALIzE SUM AND BUFF
DO 1 I=l,1500
SUM(I)=O.O
BUFF(I)=0.0
l CONTINUE
READ IN J AND IFACT
READ (60.2)J,IFACT
2 FORMAT(215) .
READ IN GAUSSIAN CURVE
READ (60.2)(YSTAR(I),I=1,183)
3.FORMAT(16F5.O)
DO 50 I=l,l83
YGAUSS(I) = YSTAR(I)
50 CONTINUE
XMaFLOAT(J)*110.O
POSITION OF FIRST PEAK
XKD=(1.05681u7-O.OO21011*XM**O.555)**3
XKDI=XKD*1000.0
NCEL=1500-IFIX(XKDI)
K=NCEL-9l
NOCEL=NCEL
APPROPRIATE FACTOR FOR GAUSSIAN CURVE
IF(IFACT,EQ,1)GOTOA
7 DO 10 I=l,183
YGAUSS(I)=FLOAT(IFACT)*YSTAR(I)
10 CONTINUE
8 GOTOA
FILL BUFF ARRAY
A DO 5 I=l,l83
BUFF(K-l+1)=YGAUSS(I)
5 CONTINUE
FILLS IN SUM ARRAY AND REINITIATES BUFF
N=K+182
DO 6 I=K,N
SUM(I)=SUM(I)+BUFF(I)
BUFF(I)=0.0
6 CONTINUE
CALCULATES NEW PEAK POSITION
1N5
PROGRAM
145
50
55
6O
65
70
3O
31
1116
SIZSIM
J=J+l
IF(J,GT,lu6)GOTO3O
XM=FLOAT(J)*110.0
XKD=(1.0568137-0.0021011*XM**0.555)**3
XKDI=XKD*lOO0.0
NCEL=1500-IFIX(XKDI)
INCK=NCEL-NOCEL
K=K+INCK
NOCEL=NCEL
DETERMINES APPROPRIATE FACTOR FOR GAUSSIAN
IOLFAC=IFACT
IP