374 KICllAHl) SCIIWKKT AND JOHN HISHOP 



Tilt' lahi'linu; of liciuo^lohin in rcticulocytt's has been studied by a 

 miinbt'i- of workers, with lathci- coullictin^ results. Muir et al. (1952) 

 injected rats with C"-valine and deinoiistrated that the specific activity 

 of the N-terniinal valine of isolated hemoglobin was the same as the 

 specific activity of the valine of the remainder of the molecule. On the 

 other hand, Ki'uh ct a/. (1907, 19()()) usino; radioactive glycine and 

 phenylalanine have obserN-ed unef|ual labelin*^ of hemoj.!;lobin in isolated 

 rabbit I'eticulocytes and in the livini;; rabbit. At times after the addition 

 or injection of the labeled amino acid ranging from 1 to 4 hours in ex- 

 periments with isolated cells, and from 5 to 58 days in the living animal, 

 hemoglobin was isolated. The specific activity of the labeled amino 

 acid released after a brief hydrolysis of each sam])lc with HCl was 

 compared with the specific activity after complete hydrolysis. In the 

 experiments with isolated cells, unequal labeling could still be detected 

 after 4 hours, while in the living rabbit uniform labeling was not 

 attained until between 30 and 50 days following injection. These experi- 

 ments are difficult to understand in terms of any of the hypotheses 

 already put forward. Since the time for synthesis of the hemoglobin 

 molecule is estimated to be approximately 1 minute (Dintzis et al., 

 1958; Schweet et al., 1961), ordered synthesis could not result in signifi- 

 cantly unequal labeling at times exceeding 20 minutes. Schapira et nl. 

 (1960) have examined the question in another way. After incubation of 

 reticulocytes with radioactive arginine, hemoglobin was isolated and 

 digested with tiypsin. The digest was "fingerprinted," the arginine pep- 

 tides identified, eluted, and hydrolyzed. The specific activity of the 

 arginine in each of nine peptides was found to be different. It should be 

 noted that Jonxis (1958) has reported four arginine residues per half- 

 molecule of hemoglobin. It appears unlikely that the long-term unequal 

 labeling was due to an analytical artifact since unequal labeling was 

 detected in two different ways. It is possible that slow, long-term 

 exchange reactions, or the presence of several types of hemoglobin with 

 different turnover rates, could explain the data. Further work is needed 

 to clarify these results. 



2. Evidence for Ordered Peptide Bond Formation 



As mentioned earlier, labeled amino acids rapidly appear in ribosomes 

 in alkali-stable linkage, presumably protein (Hoagland, 1960). In intact 

 reticulocytes, "pulse-labeling" experiments demonstrated the transitory 

 nature of this material and indicated that hemoglobin precursors, rather 

 than the structural protein of ribosomes, were formed (Dintzis et al., 

 1958; Rabinowitz and Olson, 1959). These results suggested the presence 

 of peptides in the ribosome which were hemoglobin precursors. Similar 



