Synthesis of Hemoglobin in a Cell-free System

The incorporation of Ci4-amino acids into protein, with cellfree systems containing ribosomes and soluble enzymes, has been studied extensively since the first reports by Zamecnik and Keller (1).This topic has been reviewed in detail by Hoagland (2).One limitation with many of the cell-free systems has been the failure to find incorporation into known proteins.Incorporation of CY-amino acids into ribosome-bound serum albumin (3) and soluble hemoglobin (4) was reported in 1958.More recently, incorporation into albumin (5-7), silk fibroin (8), and /3-galactosidase (9) has been observed with similar cell-free systems.Incorporation of CF-amino acids into protein in the cellfree system from rabbit reticulocytes appears to correspond to a limited synthesis of hemoglobin.Evidence for this conclusion was the stimulation of incorporation by a mixture of unlabeled amino acids (4), the agreement between the ratios of incorporation of labeled valine, leucine, and isoleucine and the amounts of these amino acids in crude globin (4), the species specificity of hemoglobin labeling when ribosomes from either mouse or rabbit reticulocytes were used (lo-12), and the comparison of NHz-terminal and internal Cr4-valine labeling (13).Detailed studies of the incorporation of C14-amino acids into protein with ribosomes and partially purified enzymes from rabbit reticulocytes will be presented here.The incorporation of Cr4-amino acids into protein, starting with amino acyl-ribonucleic acid and highly purified transfer enzyme fractions' ( 14), and studies of the final stages of completion and release of soluble protein from the ribosomez (15) will be reported in future papers.