Nascent Globin Chains from Rabbit Reticulocyte Ribosomes

Evidence is presented to show that rabbit reticulocyte ribosomes contain a significant component of completed Lu-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 a-globin pool present in reticulocytes is not a significant factor in these results. Some 4.6% of the nascent cY-globin chains are present as cy-globin-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 P-globyl-tRNA comprises 0.69% of the nascent fi-globin chains. This value coincides closely with the predicted value for nascent fl-globin chains uniformly distributed in size along the polysome. Further evidence is presented to show that both cu-globyltRNA and /3-globyl-tRNA exhibit the kinetic properties expected for normal intermediates of soluble hemoglobin biosynthesis following inhibition of the initiation of protein synthesis by pactamycin.

is presented to show that rabbit reticulocyte ribosomes contain a significant component of completed Lu-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 a-globin pool present in reticulocytes is not a significant factor in these results. Some 4.6% of the nascent cY-globin chains are present as cy-globin-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 P-globyl-tRNA comprises 0.69% of the nascent fi-globin chains. This value coincides closely with the predicted value for nascent fl-globin chains uniformly distributed in size along the polysome. Further evidence is presented to show that both cu-globyl-tRNA and /3-globyl-tRNA exhibit the kinetic properties expected for normal intermediates of soluble hemoglobin biosynthesis following inhibition of the initiation of protein synthesis by pactamycin.
This paper is an extension of previous studies of the peptidyl-tRNA fraction from rabbit reticulocytes which was reported from this laboratory (1). Those studies provided preliminary evidence of t,he existence of nascent peptides attached to tRNA whose primary amino acid sequences are those of the completed globin chains (referred to as globyl-tRYA) . While t.he peptidyl-tRNA fraction contains nascent peptides of many sizes attached to tRP;A the presence of globyl-tRNA is of particular interest since these would be expected to be the substrates for the termination and release process of globin biosynthesis.
The earlier report described a procedure for the purification of the peptidyl-tRNA component from rabbit reticulocyte ribosomes which is particularly effective in removing soluble hemoglobin contamination from the peptidyl-tRNA preparation (1). The availability of this methodology has made feasible analyses directed at the study of the globyl-tRNA molecules. The LY-and P-globin chains of rabbit hemoglobin each contain 3 tyrosine residues in their amino acid sequence (2). The COOH-terminal ends of (Y-and P-globin molecules consist of the amino acid sequences -Lys-Tyr-Arg and -Lys-Tyr-His, respectively. Since the biosynthesis of hemoglobin is known to proceed from the NHz-terminal end toward the COOH-terminal end (3) an analysis of the purified peptidyl-tRNh fraction for the presence of the COOH-terminal dipeptides tyrosyl arginine and tyrosyl histidine, following tryptic digestion, has permitted a determination of the amounts of a-globyl-tRN-4 and P-globyl-tRNA in that fraction.
While fi-globyl-tRKA exists to the extent predicted by a uniform distribution of sizes of nascent ,&globin peptides the a-globyl-tRXA 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.
Analyses of the LY-and P-peptidyl-tRNA obt,ained from uniformly labeled reticulocyte ribosomes following inhibition of further initiation with the antibiotic pactamycin indicates that the globyl-tRNX components of peptidyl-tRNA possess the kinetic properties expected for ribosomal intermediates in the biosynthesis of soluble hemoglobin. The synthetic dipeptides, n-tyrosyl-n-arginine and n-tyrosyl-n-histidine, were prepared by Cycle Chemical, Los Angeles, Calif. All other reagents used were reagent grade.
;Ilethods-Rabbit reticulocytes were obtained as described by Slabaugh and tilorris (1). Hematocrits ranged from 12 to 16. The blood was passed through loose glass wool before the reticulocytes were washed twice at 4" in the 0.9% NaCl solution (RS) described by Lingrel and Borsook (4).
Standard Incubation Conditions-A suspension of reticulocytes was incubated in a modified medium of Lingrel and Borsook (4). 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 (4) was used except that hydroxyproline was omitted and L-asparagine was added to a final concentration of 0.55 mM in the incubation medium (5). Nonradioacive n-tyrosine was added to the isotopically labeled tyrosine to a final concentration of 0.021 mM in the incubation medium.
This concentration of L-tyrosine was used for all incubations unless otherwise indicated.
All incubations were performed at 37". After an initial 2-min warm-up period the radioactive tprosine was added to the reaction mixture. This addition of radioactivity defined zero time of incubation. The incubation was terminated by pouring the entire incubation mixture, or suitable aliquots thereof, into a 12.fold volume of ice-cold RS containing cycloheximide at a concentration of 16.5 pg per ml (0.059 mM). The cells were then collected by centrifugation and washed once more with fresh RS containing cycloheximide.
Preparation of Ribosomal Pellets-The washed reticulocytes were lysed for 10 min with 4 volumes of 2.5 mM magnesium chloride containing cycloheximide (0.09 mM) and 0.21 mM sparsomycin.
The solution was then made isotonic by the addition of 1 volume of 1.5 RI sucrose-O.15 M potassium chloride. Cell debris was removed by ccntrifugation at 20,000 X g for 30 min. 1 he supernatant solution was then centrifuged at 64,000 x g for 345 hours to obtain the radioactive ribosomal pellets. Where indicated, the ribosomal pellets were resuspended in illedium 13 (6) and reisolatcd by sedimentation as before to yield washed (two times) ribosomes.
The concentration of ribonucleoprotein was determined by measuring the absorbance at 260 nm using an absorption coefficient of 11.3 for a concentration of 1 mg per ml (7).
Preparation of Peptidyl-tRNA-Ribosomal pellets were resuspended in a small volume (approximately 1 ml) of 0.25 M sucrose containing cycloheximide (0.059 mM) and 0.14 mM sparsomycin.
The ribosomal suspension was then used to prepare peptidyl-tRNA according to the method of Slabaugh and >Iorris (1). It has been found that reduction of the urea concentration of Buffers I and II from 8.0 to 7.6 M avoids the occasional problem of crystallization of the urea solutions at 4". This modification has been employed throughout these studies. The pooled fraction containing the purified peptidyl-tRNA was reduced to a volume of approximately 1.8 ml by ultrafiltration in an Amicon cell with a UM-2 Diaflo membrane.
The concentrated sample was then dialyzed against three 1500-ml portions of deionized water, lyophilized, and stored at -21". Preparations of Globin Uniformly Labeled with [l4C]Tyrosine-Washed reticulocytes were incubated in the presence of ['"Cltyrosine as described above, except that the tyrosine concentration in the medium was 0.1 mM. Penicillin and streptomycin were added to the reaction mixture to a final concentration of 0.11 mg per ml of each. Incubations were allowed to proceed at 37" for 335 to 4 hours. The cells were washed, lysed, and the postribosomal supernatant used to prepare hemoglobin according to the method of Winterhalter et al. (8). Hemoglobin concentration was determined by the method of Austin and Drabkin (9). Globin was prepared by the cold acid-acetone method (10) and stored at -20" as a lyophilized powder.
Separation of Uniformly Labeled (Y-and P-Glob& Chains-The (Y-and @-globin chains of '%-labeled rabbit globin were separated on carboxymethyl cellulose ((X-32) columns (1 x 22 cm) with a nonlinear gradient modified from the procedure of Globin (45 mg or less) was applied to the column and eluted at a flow rate of 14 to 16 ml per hour.
The separated LY-and P-globin chains were then lyophilized and each was rechromatographed on a C&I-32 column by the same procedure in order to obtain further purification. Lyophilized samples of separated globin chains were stored at -20".
The purity of the separated a-and P-globin chains obtained in this manner was established by the addition of nonradioactive carrier globin and digestion of the mixture with trypsin at 37" for 4 hours as described below.
By this means it could be shown that the a! chain preparation contained approximately 0.827; p chain while the fl chain preparation contained approximately 1% contamination by oc-globin. Analysis of Nascent Globin Chains--The lyophilized sample of labeled pcptidyl-tRNA was resuspended in 1.0 ml of water containing 0.1 mg of pancreatic RNase, incubated at 37" for 25 mm, and then lyophilized.
After redissolving in 0.15 ml of 0.1 1\~ NaOH the material was incubated for 335 hours at 37" in order to cleave the peptidylltRN,4 ester bond. The solution was then neutralized with 1 N HCl to a pH of 5.4 to 5.6 as determined with pH indicator paper.
Purified rabbit a-and pglobin chains of known radioactivity content (uniformly labeled with [14C]tyrosine) were then added as an internal standard (3). Nonradioactive globin was added, if necessary, to give a mass of 3 to 4 mg of protein in the sample. The synthetic dipeptides, L-tyrosyl-n-arginine (crT15) and L-tyrosyl-L-histidine @T16), were added as carrier peptides (50 nmoles each) prior to tryptic digestion.
Tryptic Digestion-Tryptic digestion was carried out in 0.1% NaI-IC03 (14) at a final globin concentration of 3 to 4 mg per ml. Trypsin was added in an amount equal to 2% (w/w) of the total globin present.
After 2 hours incubation at 37", lyO (w/w) trypsin was again added and the incubation continued for an additional 2 hours. Samples were then frozen and lyophilized. Separation oj Peptides-Separation of the tyrosine-containing peptides from rabbit globin was performed by the two-dimensional method of Hunt et al. (13). Electrophoresis was con-ducted in a Gilson model D high voltage electrophorator. Tryptic peptides have been numbered according to their position of occurrence relative to the NHz-terminal end of the a-and /3-globin chains of rabbit hemoglobin (15). The appropriate areas containing the radioactive tyrosine peptides were removed from the electrophoretrogram, remaining solvents removed in ~ICGCUO, and the paper was then 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 scintillation vials and lyophilized. Counting of Radioacfivity--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 containing a single radioisotope were established by internal standardization with 3H-or W-labeled toluene of known radioactivity content (New England Nuclear, Boston, Mass.). All data are expressed as decompositions per min (dpm) as determined from the observed counts per min and the counting efficiency.
The elution of radioactive materials during column chromatography was monitored by placing 25-to 50-ml aliquots of the eluate fractions in 0.5 ml of HZO, 5 ml of Aquasol, and counting in a Nuclear Chicago, Unilux 1, liquid scintillation counter. Least squares calculations were achieved with the CDC 6500 computer of Michigan State University.

RESULTS
Puri$ed, Peptic@tRhTA Is Free of Contamination w'ifh Soluble Hemoglobin-The analyses conducted in this study 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 m-as prepared (see legend of Table  I). This mixture was then subjected to the procedure for preparatiou of peptidyl-tRNA (1). Radioactivity present in the purified peptidyl-tRNA fraction thus represents the extent of The specific radioactivity of the ribosomes remained constant for at least the next 16 min. The incorporation of radioactivity into soluble hemoglobin was linear for at least the first 20 min of incorporation.
These results are consistent with those obtained by Hunt et al. (13). The constant level of radioactivity found in the ribosomal fraction after 4 min 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 min of incubation thus possess uniform specific activity of the 6 tyrosine residues present in the nascent globin chains.
Analysis of [3H]Tyrosine-labeled Peptidyl-tRNA-Rabbit reticulocytes were incubated in a medium containing [3H]tyrosine for 10 min at 37". The ribosomal pellets obtained from the labeled reticulocytes were used to prepare the purified peptidyl-tRNA fraction.
Following the addition of W-labeled LY-and fi-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 under "Experimental Procedure." The relative specific activities CH:W ratio) of the tryptic peptides are shown in Fig. 2. The 3H:14C intercepts were calculated by the method of least squares in order to obtain a relative measurement of the number of Q( and p nascent chains present. The 3H:14C ratios observed in the COOH-terminal tryptic peptides ((rT15 and PT16) were used as a relative measurement of the number of completed LY-and P-globin chains in the peptidyl-tRNA fractions since only the completed globin chains in the population of nascent chains can yield those tryptic peptides upon hydrolysis (3).
The results of three independent experiments are shown in Table II. Each set of experimental data was analyzed as shown in Fig. 2 for Experiment I. It is apparent from these data that rabbit reticulocyte ribosomes contain a significant component of completed cr-globin which is still attached to tRNA (ol-globyl-tRNA) . Some 4.6 '% of nascent cr-globin chains are present as ar-globyl-tRNA.
On the other hand, completed @ chains attached to tRNA @-globyl-tRNA) constitute only 0.70% of the nascent P-globin chains.
Effect of Hem&-The presence of a pool of free soluble cr-globin chains has been shown to be present in the reticulocyte (17, 18). It has also been reported that this pool is decreased in size if the reticulocytes are incubated with hemin (17). In order to examine the possible effects of hemin on the accumulation of cu-globyl-tRNA 'on the ribosomes two parallel incubations of rabbit reticulocytes were performed. One incubation mixture was conducted in the usual manner (see legend of Table II) while to the other was added hemin to a concentration of 1 x 10e4 M. Analysis of the six tyrosine-containing peptides from each preparation, conducted as before, revealed that hemin addition to the incubation medium did not alter the proportion of nascent cr-or P-globin chains present as ar-globyl-tRNA or /3-globyl-tRNA, respectively, in the purified peptidyl-tRNA fraction.
Pactamycin-induced Decay of Radioactivity i n ATascent Globin Chains-The antibiotic pactamycin, at a concentration of 10-O M, has been shown to inhibit preferentially the initiation of protein synthesis in the reticulocyte (19,20). Since elongation and release of nascent globin chains are not inhibited, preparations of purified peptidyl-tRNA obtained from reticulocytes that have been exposed to pactamycin should show a progressive decrease of radioactivity in the peptidyl-tRNA fraction with an increased time of incubation.
The effects of pactamycin addition to rabbit reticulocytes The ordinate represents the 3H:W ratios obtained in Experiment I of Table II. Each tryptic peptide is positioned on the abscissa according to the position of the COOH-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 NHi-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 distribution of nascent chains on the polysome.
The 3H:W intercept has been used as a measure of the total nascent chains present and the ordinate value corresponding to cvTl5 or pT16 has been used as a measure of cu-globyl-tRNA or P-globyl-tRNA present, respectively. a The 3H:W intercepts were calculated by the method of least squares as described under "Experimental Procedure." b Observed 3H:W (of orT15 or fiT16, respectively) X lOO/calculated aH:W intercept (of or-peptides or p-peptides, respectively). c Tritium content of the tryptic peptides is expressed as total decompositions per min X 10m3 by equating 3H recoveries to observed 14C recoveries. At zero time 5 mCi of [aH]tyrosine (6053 &i per pmole) were added. After 10 min of incubation the first aliquot (10 ml) was withdrawn to serve as a control prior to pactamycin addition. After an additional 15 s, as indicated by the arrow, the incubation mixture was made 1 X 1Om6 M in pactamycin by the addition of 0.6 ml of 0.5 X W4 M pactamycin in 0.9% NaCl solution (RS). Three further lo-ml aliquots were withdrawn at the time intervals indicated in the figure. Peptidyl-tRNA was prepared from each aliquot and the total radioactivity content was determined as described under "Experimental Procedures." Hemoglobin specific activities were measured in the postribosomal supernatant fractions. whose ribosomes were in a steady state of labeling (10 min at 37') is shown in Fig. 3. The specific activity of hemoglobin in the soluble phase of the reticulocyte was found to increase very little following pactamycin addition to the incubation medium, hence the amount of contamination of the ribosomal pellets with 3H-labeled hemoglobin should be similar in each of the preparations obtained at the respective time periods after pactamycin addition. However, the radioactivity found in the purified peptidyl-tRNA fraction prepared from samples withdrawn following incubation in the presence of pactamycin decrease rapidly with time. After 5 min of incubation in the presence of pactamycin, only 8.9% of the original radioactivity remained associated with peptidyl-tRNA. The radioactivity content of each of the six tyrosine-containing tryptic peptides in each of the four samples was analyzed.
These data are presented in Fig. 4. The radioactivity present in all tyrosine-containing peptides (including o~T15 and PT16) declines precipitously after pactamycin addition. Peptides atT4 and flT4, closest to the NH&erminal portion of the respective globin chains, decline most rapidly following the addition of pactamycin. These results are consistent with completion and release of nascent peptides without initiation of new peptide chains. Fig. 5 presents the relative radioactivity content of tryptic peptides near the NHt-terminal portion (crT4, /3T4) of the nascent peptides as compared to the radioactivity present in the COOH-terminal tryptic peptides (crT15, PT16). The proportions of a-globyl-tRNA and P-globyl-tRNA in the nascent protein fractions were markedly increased with time after pactamycin addition. Since (~T15 is derived only from globyl-tRNA while otT4 is derived from both globyl-tRNA and the other tyrosine-containing ar-globyl nascent peptides as well, one can determine that by 5 min after pactamycin addition approximately 24% of the nascent cy-globin peptides are ar-globyl-tRNA. To each of the peptidyl-tRNA preparations was added [Wltyrosinelabeled cr-globin (33,800 dpm) and [Wltyrosine-labeled p-globin (34,600 dpm) as a uniformly labeled internal standard prior to tryptic digestion.
A through F present the tritium radioactivity found associated with each of the six tyrosine-containing tryptic peptides as a function of time following pactamycin addition. Values are expressed as per cent of those found in the control sample removed prior to pactamycin addition.  5. Ratios of radioactivity found in tryptic peptides from the NH%-terminal and COOH-terminal portions of the nascent protein fraction following pactamycin addition. Each ratio of radioactivity was calculated from the total tritium content (dpm) of the tryptic peptides indicated.
The four peptidyl-tRNA samples analyzed are those described in Fig. 3.
A similar calculation indicates that /3-globyl-tRNA reaches approximately 7% of the nascent @globin peptides by 5 min after pactamycin addition.
The total radioactivity content of all components decreased rapidly following pactamycin addition, however. Collectively these data are consistent with the concept that the globyl-tRNA molecules are normal intermediates of soluble hemoglobin biosynthesis.

DISCUSSION
Evidence presented in this paper indicates the existence of a heretofore undetected accumulation of a-globyl-tRNA on rab-bit reticulocyte polysomes.
In the case of fl-globyl-tRNA there is no such accumulation.
If one were to assume that the size distribution of nascent globin chains is uniform (21, 22), then the cr-globyl-tRNA might be expected to be x41 or 0.71% of the total nascent chains. In contrast to this prediction, the percentage of nascent cY-peptides present as cy-globyl-tRNA was found to be more than 6 times higher than predicted. Similar calculations for the nascent /? chains would predict >i4a or 0.69% P-globyl-tRNA in the nascent fi-peptide fraction. In this case the analytical results coincide closely with the predicted value for the nascent @peptides.
A 6.4-fold excess of ar-globyl-tRNA to fi-globyl-tRNA exists in the peptidyl-tRNA fraction (Table II).
In spite of the high ratio of cy-globyl-tRNA to /I-globyl-tRNA the over-all ratio of total nascent p chains to total nascent LY chains, as determined from the intercepts of Fig.  2, was found to be 1.04, in close agreement with the finding reported by others (12, 21, 22). Various authors have, in the past, measured the percentage of nascent globin chains in the rabbit reticulocyte polysome corresponding to completed CXglobin chains or P-globin chains. However, these experiments were carried out using preparations which contained varying levels of contaminating soluble hemoglobin. This has led to a wide range of often conflicting results.
Luppis et al. found no evidence of accumulation of completed a: or p chains on the polysome (23). Hunt et al. found equal numbers of completed cy and 0 chains attached to the polysome, with results varying from experiment to experiment.
The latter authors reported the presence of from one complete globin chain per seven nascent globin chains to one completed globin chain per 60 nascent globin chains (21). Colombo and Raglioni found an excess of cY-globin chains in a reticulocyte preparation (24). On the basis of the time required for the termination and release of globin chains Lodish and Jacobsen have predicted that 570 of the nascent globin chains on the reticulocyte ribosome should be completed LY-and ,&globin chains, both chains being present in equal proportions (25). The present paper presents evidence to show that the amount of free hemoglobin contaminating the preparations of peptidyl-tRKA used to obtain the results reported here does not contribute significantly to the observed results.
This contamination amounts to not more than 0.0307, of the labeled hemoglobin present in the original ribosomal pellet.
During the preparation of peptidyl-tRNA from radioactively labeled reticulocytes, such as the preparations used to obtain the data in Table II, the average radioactivity due to soluble hemoglobin contamination of the unfractionated ribosomal pellet amounted to 6.1 X lo6 dpm. This amount of radioactivity in the hemoglobin contaminating the ribosomal pellet will leave not more than 1800 dpm of contaminating hemoglobin in the purified peptidyl-tRNA fraction. Since the supernatant hemoglobin would be expected to be nearly uniformly labeled (3), and since tryptic digestion of globin produces six tyrosine-containing peptides, the radioactivity due to each contaminant tryptic peptide would not be expected to exceed 300 dpm. In the analyses reported in Fig. 2, tryptic peptide flT16 contained an average of 14,500 dpm. The total radioactivity in all the other tryptic peptides is even greater by at least 'i-fold or more. Contamination from soluble hemoglobin (300 dpm) would introduce an uncertainty of about 0.014% to the reported value of 0.70% for fl-globyl-tRNA (Table II). Similar considerations would introduce an uncertainty due to soluble hemoglobin contamination of approximately 0.0157, to the reported value of 4.62% for a-globyl-tRNA (Table II). The uncertainty due to contamination of the other tryptic pcp-tides is negligible. The results obtained with pactamycin reinforce this conclusion.
Figs. 4 and 5 show that the preparations of purified peptidyl-tRNA contain true intermediates of globin biosynthesis.
In particular, Fig. 5 shows quite clearly the effect of inhibiting initiation of new nascent chains. While there is a decrease in the amount of nascent protein attached to the ribosomes the effect is most pronounced among those nascent protein chains in the early stages of synthesis.
Hence, the proportion of globyl-tRNA in the nascent protein fraction is increased markedly even though the absolute quantity of globyl-tRNA is reduced. Contamination of the peptidyl-tRNA fraction by soluble labeled hemoglobin would have obscured these changes.
A consideration of the ratio of aT15:flT16 found following pactamycin addition to the reaction mixture reveals an average (rT15:PT16 ratio of 5.8 during the first 30 s after addition of the antibiotic. By 2.2 min in the presence of pactamycin that ratio has been reduced to 4.9 and reaches 2.3 after 5 min of incubation. These results are incompatible with contamination of the peptidyl-tRNA fraction with free or-globin chains which have been reported to be present in the reticulocyte (18). Accumulation of oc-globyl-tRNA to the extent of 4.6a/, of the total number of nascent oc-peptides present indicates that one ribosome in 23 of those which are actively engaged in or-globin synthesis possess an a-globyl-tRNA.
From the number of ribosomes per polysome which are synthesizing a-globin in the rabbit reticulocyte (22) one can estimate that approximately one of these polysomes in five contains an cu-globyl-tRNA.
Since or-globyl-tRNA would be expected to be the normal substrate for the release steps in oc-globin biosynthesis the observed accumulation may be a reflection of a limitation of the rate of release of completed oc-globin chains from the biosynthetic template.
The biological significance of the accumulation of cu-globyl-tRKA on rabbit reticulocyte ribosomes and its relationship to the termination and release of both a-and P-globin chains in the biosynthesis of rabbit hemoglobin are currently under study in this laboratory.