Biosynthetic Ratio of Labelled Globin Chains in Human Reticulocytes, Determined by Electrophoresis on Cellulose Acetate

Abstract

A new method for determining the biosynthetic ratio of globin chains in human reticulocytes is described. Washed red cells, enriched in reticulocytes by high speed centrifugation at 30.degree. C, are incubated for 45 min at 37.degree. C with minimum essential medium (Eagle) and 3H-leucine. Globin is prepared from hemolysate by the acid-acetone method. Globin chains are separated by electrophoresis on Cellogel at pH 6.3 in 0.04 M sodium phosphate buffer, containing 6 M urea and stained with 0.5% Coomassie Blue. The strips of cellulose acetate containing the globin chains are dissolved in 10 ml of dioxan-naphtalene scintillation fluid and counted in a liquid scintillation counter. The biosynthetic ratio of globin chains is calculated from the ratio of the total counts incorporated into the .alpha. and non-.alpha. chain zones. This method gives results in agreement with those obtained by macro-CMC [carboxy-methyl cellulose] column chromatography, but is simpler and faster. This electrophoretic method appears particularly suitable as a diagnostic tool in a variety of hematological disorders.