The synthesis of hemoglobin by rabbit reticulocytes has been studied by means of pulselabelling and chase experiments. The reticulocyte lysate was fractionated into a top layer and a ribosomal fraction. The top layer was analysed by gel filtration on columns of Sephadex 6-75. At least two intermediates which were formed after short incubations and were chased into hemoglobin were separated by gel filtration. These intermediates have been identified with a chain and globin.a Chain and globin have also been separated by chromatography on carboxymethyl-Sephadex.This method of analysis has been used to measure the rate of appearance of these intermediates in the top layer fraction and their rate of removal in chase experiments. The presence of complete a chains on polyribosomes has been confirmed. The rate of appearance of a chains on polyribosomes and their rate of removal in chase experiments have been determined. These rates appear to be identical, suggesting that the same biosynthetic process is responsible for these rates.The nature of the complete a chains present on polyribosomes has been investigated. These chains can be separated from peptidyl-tRNA chains by gel filtration in a sodium dodecyl sulphateacetate buffer. This finding suggests that the complete chains are not bound to tRNA.The experimental results support the following scheme of assembly of the hemoglobin molecule : a chains are released from polyribosomes on completion. These chains combine with /3 chains which are still being synthesized on polyribosomes and for this reason complete a chains are found on polyribosomes. a/3 Subunits are released from polyribosomes once the synthesis of
The low molecular weight iron found in the guinea pig reticulocyte has been partially characterized. On thin layer chromatography it is distinguishable from the iron complexes of a variety of nucleotides, sugars, and amino acids. On paper chromatography it comigrates with a 250-nm absorbing, orcinol-positive material. The eluted count peak contains phosphorus. Approximately 1 microgram of iron is recovered from 1 ml of hemolyzed red cells. Preparation under nitrogen improves recovery of low molecular weight iron, suggesting that the iron is in the ferrous oxidation state.
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