Gel Chromatographic Analysis of Nascent Globin Chains

A gel chromatographic analytical procedure using Bio-Gel A-OSm in 6 M guanidine HCl-0.1 M Z-mercaptoethanol has been developed and standardized. The procedure permits the molecular weight of peptides in the size range of 2 to 146 amino acids in length to be established from the distribution coefficient of the peptide on the gel column. This procedure has been applied to an analysis of the size distribution of nascent peptides from rabbit reticulocyte ribosomes. These studies indicate that the size distribution of nascent peptides is nonuniform and that translation of hemoglobin messenger RNA molecules does not proceed at a constant rate in the reticulocyte.

The assembly of the polypeptide chains of hemoglobin on rabbit reticulocyte ribosomes takes place by the sequential addition of amino acids, beginning at the NH*-terminal end of the molecule and proceeding toward the COOH-terminal end of the molecule (1). A population of polyribosomes thus contains nascent globin chains of a wide range of sizes.
Development of a gel chromatographic procedure for the fractionation of nascent globin chains has permitted a study to be conducted of the size distribution of nascent peptides from rabbit reticulocytes.
While techniques for the measurement of the average rate of polypeptide synthesis during globin synthesis are available (2, 3) determination of the relative rates of translation of specific portions of the globin messenger RNA molecules has not been feasible previously.
Since an increased time in residence of a ribosome at a particular position along the messenger RNA will result in an increased amount of the corresponding nascent chain in the population of nascent chains, measurement of the relative amounts of the different sizes of nascent chains in a population of nascent chains provides a means of accessing the relative rates of translation along the messenger RNA molecules.
Data presented here reveal that a nonuniformity of size distribution exists among the nascent globin chains. These results indicate that the rate of translation of the messenger RNA Reagents-Guanidine HCl (grade 1) was purchased from Sigma Chemical Co., St. Louis, MO., and recrystallized twice according to the procedure of Nozaki and Tanford (4). Cvcloheximide and 2-mercaptoethanol were also obtained fro& Si&a Chemical Co. Bio-Gel-A-0.5m (200 to 400 mesh, 10% agarose); Bio-Gel P-10 (50 to 100 mesh), Bio-Gel P-2 (100 to 200 mesh). and Dowex 50-X8 (100 to 200 mesh;k+ form) were purchased fro& Bio-Rad Laborator'ies, Richmond, Calif. Blue dextran 2000 (mol wt 2 X 106) and the K 15/90 chromatographic column (1.5 X 90 cm) were purchased from Pharmacia Fine Chemicals, Piscataway, N. J. Labeled amino acids were purchased from Amersham Searle Corp., Arlington Heights, Ill. Aquasol was from the New England Nuclear Corp., Boston, Mass. Cyanogen bromide was bought from Distribution Products Industries, Rochester, N. Y. Trgpsin was L-1-tosylamido-2-phenylethyl chloromethyl ketone-trt%ed, obtained from Worthington Biochemicals Corn.. Freehold. N. J. Penicillin G was fromthe Upjohn Co., Kalam'rtsoo, Mich. ' St,reptomycin sulfate (USP) was-from General Biochemicals, Chagrin Falls, 0. Carboxymethylcellulose (CM32) was from H. Reeve Angel and Co., Clifton, N. J. DNP-alanine' was provided by Dr. R. J. Evans of Michigan State University and sparsomycin was donated bv Drug Research and Develonment. Division of Cancer Treatment, National Cancer Institute,'Bethesda, Md. Column Preparation-Bio-Gel A-0.5m was suspended in water, allowed to settle, and the supernatant solution was decanted. This wash procedure was repeated several times. The agarose gel was then suspended in water and solid guanidine HCl was added to a final concentration of 6 M, 2-mercaptoethanol was added to 0.1 M. The mixture was degassed in vucuo and titrated to pH 6.5 with NaOH. The mixture was then equilibrated overnight at 4" and the gel slurry was added to the chromatography column using 60 cm of solvent hydrostatic pressure. Column beds, ranging in heights from 78 to 83 cm, were prepared and washed for 36 hours with 6 M guanidine HCI-0.1 M 2-mercaptoethanol, pH 6.5. Flow rates from these columns were approximately 3 ml per hour. All gel chromatography analyses were conducted at 4" and 60 cm of solvent hydrostatic pressure.
Samples of peptides to be analyzed were combined with blue dextran (0.80 mg) and DNP-alanine (25 fig) in 6 M auanidine HCl-0.1 M 2-mercaptoethanol-7.5y0 sucrose (to&l volu<e 0.21 ml), and titrated to DH 8.6 with NaOH as determined with DH-indicator paper. After standing at room temperature for seveial hours the solution was adjusted to pH 6.5 with HCl, chilled, and layered onto the top of the Bio-Gel A-0.5m column using a Sage pump (Sage Instruments, Inc., White Plains, N. Y.). Eluate fractions were collected using a Gilson linear fractionator (Gilson Medical Electronics, Middleton, Wis.) equipped with a drop counter attachment, or directly into glass scintillation vials fastened to a round fraction collector (Instrument Specialties Co., Lincoln, Neb.) using the drop counter attachment.
Eluate fractions containing the blue dextran and the DNP-alanine markers were measured spectrophotometrically at 630 and 360 nm, respectively, in a Gilford spectrophotometer, model 24005 (Gilford Instrument Laboratories, Inc., Oberlin, 0.). Counting of radioactivity in the eluate fractions was accomplished using a Unilux I Nuclear Chicago liquid scintillation counter (Nuclear Chicago, Des Plaines, Ill.) for those samples containing a single isotope or using a Packard model 3310 liquid scintillation spectrometer for samples containing two isotopes. Aquasol (5 ml) was added to each aliquot or sample counted.
The decompositions per min (dpm) in each sample were calculated by the channel ratio method.

Incubation of Rabbit
Reticulocytes-Rabbit reticulocytes were obtained as described bv Slabaueh and Morris (5). Followine an initial 2-min preincubation, to alhieve temperature equilibration, labeled amino acids were added to the reaction mixture and the incubation continued for 10 additional min at 37". This procedure has been shown to provide uniform labeling of the nascent peptides on the reticulocyte polysomes (6). Cells were washed in isotonic solutions containing 0.059 rnM cycloheximide.
Lysis of the cells and isolation of the polyribosomes was conducted in solutions containing 0.059 mM cycloheximide-0.14 mM sparsomycin to prevent further peptide bond formation during polysome isolation procedures (6). emerged and the peptide was then eluted with 4 "M pyridine and lyophilized to dryness. Preparation and Chromatography of Nascent Globin Peptides- The polysome pellets were resuspended in a small volume of 0.25 M sucrose-O.059 mM cycloheximide-0.14 mM sparsomycin, and peptidyl-tRNA was prepared by the method of Slabaugh and Morris (5). The urea concentration of Buffers I and II of this procedure was reduced to 7.6 M to avoid possible problems of crystallization of the urea-containing solutions during the preparative procedures at 4". The pooled fractions of the Buffer II eluate containing peptidyl-tRNA were reduced in volume to about 1.8 ml by ultrafiltration in an Amicon cell with a UM-2 Diaflo membrane (Amicon Corp., Lexington, Mass.). The concentrated sample was then dialyzed against three 1509ml portions of deionized water, lyophilized, and stored at -20".
Each of the larger (cyanogen bromide) peptides was hydrolyzed in vucuo with 6 N HCl at 110" for 22 hours. The hydrolysates were subjected to automated amino acid analysis.
Smaller (tryptic) peptides were characterized by paper electrophoresis at pH 4.7 on Whatman No. 3MM paper according to the method of Hunt et al.
(2). Labeled tyrosine-containing peptides were located using a Packard model 7201 radiochromatogram scanner equipped with a model 385 recording ratemeter.
A tryptic digest of %-labeled p-globin was run in parallel as a reference standard and tyrosinecontaining peptides were visualized using a I-nitroso-2-naphthol stain (14).

Construction of Theoretical Elution
Patterns of Nascent Globin Chains-Since it was experimentally impractical to introduce labeled amino acids into all amino acids of the nascent elobin fraction it became necessary to construct theoretical elution curves to serve as a reference with which to compare the observed elution profiles for peptides labeled with one amino acid. These elution patterns were-calculated using the following assumntions. 1. The size distribution of nascent peptides is uniform. 2. The elution pattern of anv single nentide will be a Gaussian Prior to addition of the nascent peptides to the Bio-Gel A-0.5m column the peptidyl-tRNA was dissolved in 0.5 ml of 0.1 N NaOH and incubated 3% hours at 37" in order to hydrolyze the peptide to tRNA ester bond. The solution was then neutralized with 1 N HCl to pH 8.0 to 8.4 as determined with pa-indicator paper. The sample was then lyophilized and prepared for analysis as indicated under "Column Preparation." curve. The Gaussian curve w>ll be centered at the distribution coefficient (Kd) value of its corresponding peptide.
The distribution profile of each peptide is independent of other peptides.
3. The ratio of nascent @-globin chains to nascent 01 chains is 1. (Hunt et al. (15) report this ratio to be 1.1 while our own studies yielded a ratio of 1.04 (6).)

Preparation of Tyrosine-labeled Globin
Chains-Washed reticulocytes were incubated in the presence of ['"Cltyrosine or [3H]tyrosine according to the method of Lingrel and Borsook (7) as modified by Hunt (8). The final concentration of tyrosine in the reaction mixture was 0.1 mM. Penicillin and streptomycin were each included in the reaction mixture at 0.11 mg per ml. The reticulocytes were incubated 3% to 4 hours at 37", washed, lysed, and the postribosomal supernatant used to prepare hemoglobin as described by Winterhalter and Huehns (9): Hemoglobin concentration was determined bv the method of Austin and Drabkin (10). Globin was prepared by the cold acid-acetone method of Rossi-Fanelli et al. (11). The labeled a-globin and fl-globin chains were separated on carboxymethylcellulose (CM32) as described previously (6), lyophilized, and stored at -20".
4. For a population of nascent peptides labeled with a single amino acid, the area under the elution pattern of any one peptide will be proportional to the number of residues of labeled amino acid present.
5. Molecular weight increases in steps of 110, the average molecular weight of amino acids of /3-globin. Nascent a-globin chains and nascent b-globin chains having the same number of residues will have the same distribution coefficient. The relationship between the distribution coefficient of each nascent peptide employed in the construction curve was determined from the experimental relationship defined under "Results" (Fig. 2). The construction of the theoretical elution pattern consists of drawing Gaussian curves for each of 145 peptides sizes and adding the ordinates of the resulting curves at each Kd value. The theoretical elution patterns reported here were determined using the CDC 6500 computer of Michigan State University.

Preparation of Cyanogen
Bromide Fragments of Globin-The unfractionated tyrosine-labeled 01-and fl-globin mixture was dissolved in 70yo formic acid (v/v) to a final concentration of 5 mg per ml. A 400-fold molar excess of CNBr was then added and the mixture was stirred at room temperature in the dark for 48 hours. Ten volumes of water were then added and the solution was lyophilized to dryness (12).
Preparation of Tyrosine-labeled Tryptic Peptides-The tyrosinelabeled tryptic peptides of @-globin were prepared by digestion of the purified fl-globin chain (0.3 mg per ml) with 1.3% (w/w) trypsin in 0.2 M ammonium bicarbonate for 10 hours at 37". The sample was then lyophilized, redissolved in a small volume of 0.2 M ammonium bicarbonate and passed through a Bio-Gel P-10 column (1 X 25 cm) equilibrated with 0.2 M ammonium bicarbonate in order to separate the remaining trypsin and undigested globin from the tryptic peptides.
Aliquots (50 ~1) from eluate fractions (3 ml each) were analyzed for radioactivity by liquid scintillation counting using Bray's solution (13). The void volume fractions containing trypsin and undigested p-globin (7 through 11) were discarded and the fractions containing the labeled tryptic peptides (18 through 33) were pooled and lyophilized. Fractions-Removal of guanidine HCl and 2-mercaptoethanol from the eluate fractions prior to characterization of the properties of the eluated peptides was accomplished by gel filtration of the samples on Bio-Gel P-2 columns equilibrated and eluted with 0.2 M ammonium bicarbonate. The eluted peptides were then lyophilized to dryness. Removal of guanidine HCl and 2-mercaptoethanol from the fractions containing the smallest peptide (gT16) was accomplished bv adding the sample to a Dowex 50 column (1.1 X 40 cm) equilibrated with 0.1 M pyridine.
The column was washed until the auanidine HCl RESULTS Nascent peptides of rabbit globin contained on a population of reticulocyte polysomes would be expected to contain nascent globin chains in all degrees of completion.
Thus, this population of peptides should range in length from a minimum of 2 amino acids to a maximum of 146 amino acid residues. This prediction may be deduced from the NHz-to COOH-terminal direction of synthesis of globin chains during the translation of the hemoglobin messenger RNA molecules (1) and from the amino acid sequence of rabbit hemoglobin (17). In seeking to develop an analytical method which would allow accurate measurement of the full range of size distribution of the nascent peptides, a study was conducted of the properties of a Bio-Gel A-0.5m column eluted with 6 M guanidine HCl-0.1 M 2-mercap-toethanol in resolving small peptides.
Use of the dense agarose E matches the composition of the 55 amino acid-containing gel, Bio-Gel A-0.5m, for the fractionation of peptides smaller cyanogen bromide fragment of P-globin (PCB-1). Particularly than 20,000 molecular weight was suggested by the data of noticeable is the absence of isoleucine.
Peptides D and C are Fish et al. (16) who utilized the less dense Bio-Gel A-5m (6% resolved incompletely from one another and therefore the agarose) to fractionate polypeptides having molecular weights amino acid composition of the peptides analyzed should show of from 1,540 to 76,600.
only a degree of enrichment of one peptide over another. The Radioactively labeled peptides of known size were prepared results obtained are consistent with the designation of Peptide by cyanogen bromide cleavage of tyrosine-labeled rabbit cr-glo-D as PCB-2 and Peptide C as aCB-2. This conclusion is bin and P-globin and by tryptic hydrolysis of tyrosine-labeled further substantiated by the elution profile obtained from a P-globin.
Since each globin chain contains 1 methionine residue, cyanogen bromide cleavage yields 4 peptides ranging in molecular weight from 3,413 to 11,996. Each globin chain contains 3 tyrosine residues. Tryptic digestion of the tyrosinelabeled fl-globin chain yields 3 radioactive tyrosine-containing tryptic peptides of molecular weight ranging from 316 to 1,526. Procedures." Radioactivity found in the @-globin were combined with 5.4 mg of [SH]tyrosine-labeled globin eluate fractions is shown in Fig. 1. Seven major peaks of (see Table I). Blue dextran and DNP-alanine were added, the radioactivity are apparent in the elution diagram. The wide mixture was applied to a Bio-Gel A-0.5m column and eluted with peak of radioactivity seen in Fractions 102 to 117 was shown 6 M guanidine HCl-0.1 M 2-mercaptoethanol (see "Experimental Procedures" for details).
Aliquots (15 ~1) of eluate fractions (0.8 to consist of two peptides which elute from the column near to ml each) were analyzed for SH and 1% content.
Selected eluate one another (see inset, Fig. 1). Eluate fractions were pooled fractions were pooled (horizontal bars) and lyophilieed following and the guanidine HCl and 2-mercaptoethanol were removed as removal of guanidine HCI and 2-mercaptoethanol (see "Experidescribed under "Experimental Procedures." Amino acid anal-mental Procedures"). Each lyophilized sample was analyzed to ysis of Peaks C, D, E, and F are reported in Table II. Pep-establish the identity of the peptide contained (see text). Inset, independent analysis of a mixture containing [Wltyrosine-labeled tide F matches very closely in amino acid composition the 32 P-globin and peptides obtained following cyanogen bromide cleavamino acid-containing cyanogen bromide fragment of cY-globin age of [3H]tyrosine-labeled cu-globin and [Wltyrosine-labeled p-(&B-l). Particularly characteristic is the absence of phen-globin.
ylalanine and the low leucine content. The presence of the homoserine produced by the action of cyanogen bromide is sug-  position of the peptides indicated (see Table I).
The smaller peptides were characterized on the basis of their elect.rophoretic mob&ties at pH 4.7 using the known mobilities of the tryptic peptides of fl-globin as the reference standard (2). These results (not shown) indicate Peptide G is the 12 amino acid-containing Peptide PT14, Peptide H is the 10 amino acidcontaining Peptide flT4, and Peptide I is the dipeptide PT16.
Gel chromatographic analysis of 3H-labeled tryptic peptides of P-globin had previously shown that no radioactivity was eluted in the fractions near the blue dextran marker.
Since high molecular weight materials were removed from the tryptic digest,ion mixture with P-10 chromatography (see "Experimental Procedures") the peak of 3H-labeled material eluting in Fractions 90 to 99 was deduced to be the added 3H-labeled P-globin.
Treatment oj Data-Fish et al. (16), using Bio-Gel A-5m, studied the relationship between the molecular weight of peptides in the range of 76,000 to 1,540 and the relative positions of those peptides in the elution diagram.
Using blue dextran and DNP-alanine as reference markers, a plot of the cube root of the distribution coefficient (Kd) of the polypeptide versus the molecular weight of that peptide raised to the 0.555 power yielded a straight line relationship.
A similar treatment of data reported here using Bio-Gel A-0.5m and peptides in the molecular weight range 16,003 to 316 is shown in Fig. 2. It may be seen that the relationship between KJ/3 and (molecular weight)0'55j remains linear throughout the molecular weight range of the materials obtained from labeled rabbit hemoglobin.
The studies which follow report the application of this gel filtration technique, using Bio-Gel A-0.5m, to the investigation of the size distribution of nascent globin chains of rabbit reticuocyte ribosomes.
Analysis of Size Distribution of [3H]Tyrosine-labeled Nascent Globin Chains-Tyrosine residues occur in the amino acid sequence of cu-globin chain of rabbit hemoglobin at positions 24, 42, and 140 (17). The fl-globin chain contains tyrosine at residues 35, 130, and 145. On the premise that a uniform size distribution of (Y-and @globin chains exist in the nascent globin fraction one can construct a theoretical elution pattern for tyrosine-labeled nascent globin peptides, which are uniformly labeled, on the basis of the relationships between molecular weight and Kd described in Fig. 2. The elution profile shown in Fig. 3 Table I. Distribution coefficients were calculated from the relative positions of elution of peptides between the position of elution of the blue dextran and DNP-alanine markers ---nascent globin peptides which possess a uniform distribution of peptide sizes. The pattern reflects the positions of incorporation of tyrosine during hemoglobin synthesis. The properties of the Bio-Gel A-0.5m column in fractionating a mixture of peptides of different sizes results in a smaller increase in Kd value between any one peptide and the peptide which is one amino acid longer as the size of the peptides increase. Consequently an eluate fraction collected at Kd = 0.3 would be expected to contain more members of the nascent peptide population than an eluate fraction of the same volume collected, for example, at Kd = 0.6. This latter feature results in a theoretical elution profile having an increasing slope with decreasing Kd.
The elution profile observed when a sample of [aH]tyrosinelabeled nascent globin peptides was analyzed by gel chromatography on Bio-Gel A-0.5m is shown in Fig. 4. While the observed elution profile possesses the general dimensions predicted in Fig. 3, several unexpected features are present.
Most noteable of these is the presence of "peaks" and "troughs" which are apparent in the profile.
In a uniformly labeled population of nascent peptides these "peaks" and "troughs" The radioactive materials which elute from the column near the blue dextran marker have been found in all analyses conducted to date. Since digestion of the peptidyl-tRNA preparation with pancreatic ribonuclease, prior to alkaline hydrolysis of the peptide to tRNA ester bond, does not change this component one may conclude that the material is not unhydrolyzed peptidyl-tRNA.
Conditions under which samples are prepared for gel chromatography (6 M guanidine-0.1 M 2-mercaptoethanol at room temperature for several hours) tend to rule out aggregation due to disulfide bond formation.
While the exact nature of this component has not been established Lodish (19) has recently shown that soluble reticulocyte ribosomes synthesize small amounts of high molecular weight membrane proteins. The nascent peptides of these proteins may be present in this peak of radioactivity.
Analysis of Size Distribution of [3H]Tryptophan-labeled Nascent Globin Chains-Tryptophan residues are incorporated early during globin biosynthesis since they occur at position 14 of the cr-globin chain and 15 and 37 of the P-globin chain.
Consequently, the theoretical elution pattern (Fig. 5) is less complex for tryptophan-labeled peptides. Data obtained from Bio-Gel A-0.5m chromatographic analysis of [aH]tryptophan-labeled nascent peptides is shown in Fig. 6. The elution profile contains the peaks and troughs noted earlier with [aH]tyrosinelabeled peptides (Fig. 4).
Size Distribution of Nascent Peptides Prepared from Fresh Whole Blood-Tepper and Wierenga (20) have reported that rabbit reticulocytes maintained in the cold rapidly lose their polyribosomal structures.
These polyribosomes re-form following incubation at 37" for 2 min. These authors noted an oscillatory rate of hemoglobin biosynthesis in rabbit reticulocytes following chilling, presumably due to synchronous initiation of globin chains during that period.
Reticulocytes kept at 37" prior to incubation in the presence of labeled amino acids synthesize hemoglobin at a constant rate.
In order to examine .whether the results observed during Bio-Gel A-0.5m chromatography were in some way related to the chilling of reticulocytes during isolation and washing of the cells prior to incubation, an analysis of [3H]tryptophan-labeled nascent peptides from reticulocytes labeled in unchilled whole blood was conducted. Fig. 7 shows that the size distribution of the nascent globin peptides is unrelated to the temperature during reticulocyte preparation.
Size Distribution of [a5S]Methionine-labeled Nascent Peptides-While each globin chain contains only 1 methionine in its final amino acid sequence (at position 32 of cr-globin and position 55 of /3-globin) the initiation of globin biosynthesis has been reported to involve insertion, and subsequent removal, of an NH*-terminal methionine residue (21)(22)(23)(24). Fig. 8 presents the results of an analysis of [35S]methionine-labeled nascent peptides.
From Fig. 2   tryptophan-labeled nascent globin peptides from reticulocytes of unchilled whole blood. Blood (40 ml) was drawn from an anemic rabbit by heart puncture, filtered through glass wool, and incubated immediately with 3 mCi of VIltryptophan (specific activity 7.1 mCi per pmole) for a period of 12 min at 37". The reaction was terminated and peptidyl-tRNA prepared as before (see "Experimental Procedures"). residues should first appear in cr-globin peptides which elute at Kd = 0.65 and in P-globin peptides which elute at Kd = 0.52. Radioactivity associated with peptides eluting after Kd 0.65 must arise from those peptides containing NHz-terminal ['%met.hionine.
One would also expect that some of the labeled material at Kd = 1.0 is free methionine liberated from methionyl-tRNA during alkaline hydrolysis of the peptidyl-tRNA fraction since some amino acyl-tRNA would be expected to be present.
No theoretical elution profile for methionine-labeled peptides was constructed due to the uncertain contribution of the NHz-terminal to the expected profile. The peaks and troughs observed in the elution profile of [35S]methionine-labeled nascent peptideS may be seen to resemble closely those obtained with [3H]tyrosine (Fig. 4)  Our modification of the latter procedure using the more concentrated agarose gel Bio-Gel A-0.5m provided an orderly resolution of peptides in the molecular weight range from 316 (dipeptide) through 16,000 (146 amino acids) and hence provides a useful method for the analysis of the size distribution of nascent globin chains which would be expected to span that particular range of molecular weight values.
Several workers have attempted to assess the size distribution of the nascent globin fraction using the specific activity of tryptic peptides of nascent chains as a measure of uniformity or nonuniformity (15,(26)(27)(28). While earlier studies indicated nonuniformity to exist among the nascent globin population more recent work has indicated quite the opposite, suggesting that the size distribution was, indeed, uniform. The analysis of tryptic peptide specific activities has been shown to reveal nonuniform size distribution in instances of severe amino acid starvat,ion or inhibition of initiation of protein synthesis by NaF (15). The ability of this procedure to detect more subtle deviations from uniformity may be questioned, however.
Since each tryptic peptide analysis is obtained from a wide range of nascent peptide sizes the method seems inherently not well suited for the detection of nonuniformity of size distribution.
The gel chromatographic method reported here for the analysis of labeled nascent peptides from rabbit reticulocyte ribosomes possesses the advantage of analyzing the size distribution of the nascent peptides more directly.
While the labeled materials eluted from the Bio-Gel A-0.5m column would be expected to consist of a composite of the nascent peptides of cr-globin, P-globin, and the non-globin proteins which are synthesized by the rabbit reticulocyte (19), Woodward et al. (29) have demonstrated that less than 57, of the [aH]tyrosine-or [aH]tryptophanlabeled proteins synthesized by these reticulocytes are nonglobin proteins.
Consequently, the elution patterns obtained with gel chromatography must reflect, almost entirely, the size distribution of nascent globin peptides. These studies indicate that the size distribution of nascent globin peptides is not uniform but that certain sizes of nascent chains predominate in the pattern while others appear in a much lesser frequency.
While the elution profiles reported here are complex, the accumulation of nascent peptides of approximately 40, 57, and 89 amino acids in length may be recognized as consistent features of the nascent peptide population.
In addition, these data suggest that an accumulation of peptides in the 120 to 145 amino acid size range may be present.
The accumulation of the completed cr-globin chain, still attached to tRNA (cy-globyl-tRNA), on rabbit reticulocyte ribosomes has been reported earlier (6).
Since the length of a nascent peptide is indicative of the relative position of the ribosome along the messenger RNA during the course of translation one may conclude that at any one instant ribosomes are more apt to reside at certain regions of the messenger RNA strand than at others.
We interpret these data to indicate that the rate of translation of the globin messenger is not constant but proceeds more rapidly in some regions (hence producing the observed troughs) and more slowly in other regions (producing the observed peaks). These data also indicate that more than one slow region is encountered during translation.
The etiology of variations in the translation rate of the hemoglobin messenger RNA molecules presents an intriguing subject for future investigations.