A TECHNIQUE FOR ESTIMATION OF HEMOGLOBIN BIOSYNTHESIS (β/α RATIO) BY ISOELECTRIC FOCUSING ON AMPHOLINE-POLYACRYLAMIDE GEL
A new method for estimation of the (β/α) globin chain ratio of hemoglobin biosynthesized in reticulocytes incubated in a culture medium containing radioisotopic leucine is described. Carbonmonoxyhemoglobinized hemolysate prepared from the reticulocytes was treated with PCMB solution, and subjected to isoelectric focusing on an ampholine-polyacrylamide gel plate to separate the PMB -α and the PMB-β hemoglobins. They were separately eluted into phosphate buffer solutions, their heme was removed with acid-acetone, and dissolved in Triton X-100 solutions. The radioactivities (dpm) of the solutions of heme-free PMB-α and PMB-β were counted. Their specific activities (dpm ÷ absorbance of the eluates at 415 nm), α andβ, were calculated to get the β/α ratio of the biosynthesized hemoglobin. The analytical data obtained about the normal subjects and the β-thalassemia subjects by this method were in good agreement with those obtained by the standard method of Clegg-Naughton-Weatherall.