Abstract
Abstract The incorporation of labeled amino acids into the α and β chains of hemoglobin has been studied in rabbit reticulocytes. Relative rates of synthesis were assessed by measurement of the specific activities of globin and of constituent α and β chains prepared from unpurified, ribosomefree hemolysates. The α:β specific activity ratio of approximately 1.0 indicated that labeled α and β chains were produced in nearly equal numbers. However, in hemoglobin which was purified from these hemolysates two features were noted that indirectly suggested the presence of a pool of α chains and a smaller pool of β chains: (a) the α:β specific activity ratio was less than 1.0, and (b) a loss of α chain radioactivity and a smaller loss of β chain radioactivity were observed following purification of the hemoglobin. More direct evidence for these previously formed pools has been found in (a) the radioactivity elution pattern observed on gel filtration of unpurified hemolysate, which revealed a radioactive protein of high specific activity, and (b) further analysis of this minor peak by ion exchange chromatography, peptide mapping, and spectrophotometry. Incubation with added hemin (1 x 10-4 m) was associated with (a) an increase in the specific activities of globin and of α and β chains, (b) the disappearance of the pool of β chains, which probably existed as αβ dimers, (c) persistence of a pool of α chains during the periods of incubation used, and (d) an increase in the α:β specific activity ratio in purified hemoglobin. A model of hemoglobin biosynthesis is suggested which could account for the observed effects of added hemin in terms of (a) stimulation of the synthesis of α and β chains, (b) combination of hemin with αβ dimers (globin) to form hemoglobin, and (c) promotion of the assembly of newly synthesized α and β chains, thus largely bypassing the pool of α chains. In this manner, heme may be said to coordinate as well as stimulate the synthesis of hemoglobin.
Highlights
Incubation with added hemin (1 X 10M4~) was associated with (a) an increase in the specific activities of globin and of CYand 6 chains, (b) the disappearance of the pool of /3 chains, which probably existed as cr/3 dimers, (c) persistence of a pool of CYchains during the periods of incubation used, and (d) an increase in the ol:p specific activity ratio in purified hemoglobin
A model of hemoglobin biosynthesis is suggested which could account for the observed effects of added hemin in terms of (a) stimulation of the synthesis of a and fi
Effect of added hemin on relative specijic activities of o1 and fl chains of globin prepared from unpurijied by carboxymethyl cellulose chromatography In Experiments 1 and 2, the incubation was carried out with reticuloeytes produced by bleeding deficient diet; Experiments 3 and 4 utilized reticulocytes from APH-treated rabbits
Summary
Method of purificationDecrease in specific activity - Percentage decrease in specific activity ._Globin cc Control 4. With allowance for the different amounts of material loaded onto the columns, it was calculated that 85% of the minor peak from the original hemin-treated cells (Fig. 6B) was recovered from CMcellulose-purified hemoglobin by Sephadex G-100 gel filtration (Fig. 6D). These findings lead to the conclusion that the minor peak separable on Sephadex G-100 is included in part (control) or almost wholly (hemin-treated) in the main hemoglobin eluates of the CM-cellulose purification column. Still unresolved are the questions of the relationship between the existence of a monomer peak and the possible effect of CM-cellulose on the stability of dimers, and the possibility that exchange of heme between hemoglobin and nonheme-containing subunits may have contributed to the presence of 410 rnp absorbance in the minor peaks [19, 20]
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