Quantitation of hemoglobin biosynthesis with agarose isoelectric focusing
作者:
Anne G. Leary,
Pamela N. Porter,
Makio Ogawa,
期刊:
American Journal of Hematology
(WILEY Available online 1983)
卷期:
Volume 14,
issue 1
页码: 67-73
ISSN:0361-8609
年代: 1983
DOI:10.1002/ajh.2830140108
出版商: Wiley Subscription Services, Inc., A Wiley Company
关键词: hemoglobin biosynthesis;isoelectric focusing;agarose
数据来源: WILEY
摘要:
AbstractIsoelectric 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.
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