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°C, are incubated for 45 min. at 37°C with Minimum Essential Medium (Eagle) and ³H-Leucine.

Globin is prepared from hemolysate by acid-acetone method. Globin chains are separated by electrophoresis on “Cellogel” at pH 6.3 in 0.04 M sodium phosphate buffer, containing urea 6 M, 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 α and non- α chain zones.

This method gives results in agreement with those obtained by macro-CMC column chromatography, but it is much simpler and faster. This electrophoretic method appears particularly suitable as a diagnostic tool in a variety of hematological disorders.