MEASUREMENT OF HEMOGLOBIN CHAINS BOUND TO THE ERYTHROCYTE-MEMBRANE - DEVELOPMENT OF A METHOD AND STUDIES OF INCUBATED NORMAL-CELLS

  • 1 January 1980
    • journal article
    • research article
    • Vol. 96  (2) , 318-327
Abstract
A method utilizing sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of the .alpha. and .beta. chains of Hb was adapted to quantify Hb chains by simultaneous electrophoresis of globin standards with unknowns, staining with Coomassie blue, densitometric scanning and planimetry. This method was used to quantify the globin chains bound to the human red cell membrane during sterile incubation and ATP depletion in vitro. The following observations were obtained in thoroughly washed (6-step) ghosts of incubated cells: more .beta. and .alpha. chains were bound (.beta./.alpha. ratio 1.28); only about 1/3 of the bound chains contained heme; and globin accounted for less than 20% of the excess protein present in the incubated cells compared to fresh cells. Four-step ghosts contained more Hb, a smaller proportion of heme-free .alpha. and .beta. chains, and approximately equal numbers of .alpha. and .beta. chains (.beta./.alpha. ratio 1.09).