Measurement of hemoglobin chains bound to the erythrocyte membrane. Development of a method and studies of incubated normal cells.

A method utilizing sodium dodecyl sulfate--polyacrylamide gel electrophoresis for the sepration of the alpha and beta chains of hemoglobin has been adapted to quantify hemoglobin chains by simultaneous electrophoresis of globin standards with unknowns, staining with Coomassie blue, densitomeric scanning, and planimetry. This method has been used to quantify the globin chains bound to the human red cell membrane during sterile incubation and ATP depletion in vitro. In thoroughly washed (6-step) ghosts of incubated cells; (1) more beta than alpha chains were bound (beta/alpha ratio 1.28); (2) only about one third of the bound chains contained heme; and (3) globin accounted for less than 20% of the "excess" protein present in the incubated cells compared to fresh cells. Four-step ghosts contained more hemoglobin, a smaller proportion of heme-free alpha and beta chains, and approximately equal numbers of alpha and beta chains (beta/alpha ratio 1.09).