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Quantitation of hemoglobin biosynthesis with agarose isoelectric focusing
Author(s) -
Leary Anne G.,
Porter Pamela N.,
Ogawa Makio
Publication year - 1983
Publication title -
american journal of hematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.456
H-Index - 105
eISSN - 1096-8652
pISSN - 0361-8609
DOI - 10.1002/ajh.2830140108
Subject(s) - isoelectric focusing , agarose , hemoglobin , isoelectric point , fetal hemoglobin , chromatography , chemistry , elution , biosynthesis , biochemistry , microbiology and biotechnology , biology , fetus , gene , genetics , enzyme , pregnancy
Isoelectric focusing in polyacrylamide gels has been widely used to separate fetal (HbF) and adult (HbA) hemoglobins, and the relative synthesis of HbA and HbF has been estimated by fluorography or autography of dried gels. In order to measure the absolute synthesis of hemoglobin, we developed a system that utilizes isoelectric focusing in agarose and quantitates the total radioactivity of separated hemoglobins. After isoelectric focusing, the protein bands are individually eluted from the agarose and the radioactivity measured by liquid scintillation counting. We used this technique to study the synthetic capabilities of erythroid precursors at sequential times in culture. As previously reported, the relative ratio of HbF decreased over time in culture. However, our results clearly revealed that the absolute synthesis of HbF did not decrease until there was a parallel decrease in hemoglobin A, and that changes in the relative ratio occur because of disproportionate increases in HbA. This methodology, allowing independent evaluation of the radioactivity in synthesized hemogrlobin, enabled us to gain new insight into the biosynthetic capabilities of erythroid precursors in clonal cell culture.