Kinetics of Ligand Binding in the Hemoglobin of Lumbricus terrestrist

Sodium dodecyl sulfate gel electrophoresis and determinations of the per cent Fe in the hemoglobin of Lumbricus terrestris indicate 1 heme/17, 000. This finding suggests that the double-hexagonal hemoglobin structure revealed by electron microscopy consists of 12 subunits, each containing 16 hemes. Gel filtration molecular weight studies on the hemoglobin of Lumbricus show a decrease in molecular weight from 2.5 X 106 at pH 7 to 0.25 X 106 (and lower) at pH 10.3. The reactions studied, CO combination and oxygen dissociation, are sensitive to changes in pH and protein concentration. Kinetic measurements strongly suggest that there is little if any cooperativity in ligand binding in the isolated 16-heme subunits. The high cooperativity in ligand binding shown by the hemoglobin from Lumbricus must arise from interactions among the 16-heme subunits.