COMPARISON OF RESISTANCE OF HUMAN AND HORSE FERRIHEMOGLOBIN LIGAND DERIVATIVES TO ACID DENATURATION

The velocity of denaturation of human ferrihemoglobin by dilute acid is greatly reduced by complexing with ligands (CN-, N3-) that form low-spin complexes with the heme iron; it is similarly, but much less strongly, affected by the formation of high-spin complexes with other ligands (formate, F-, OCN-, SCN-). The effects are similar to those previously found with horse hemoglobin, but partial stabilization by high-spin ligands is more marked in human hemoglobin. The mechanism of stabilization appears to be strengthening of the stabilizing heme protein interaction so that lower pH values are required to initiate heme separation, which seems to be required for unfolding; neutralization of the positive charge on the ferriheme also seems to be involved. Use has been made of the more stable CN- derivatives to confirm conclusions based on earlier measurements of the numbers of basic groups unmasked incident to unfolding by acid, by extending measurements to lower pH. The results confirm earlier findings that more histidines per heme are masked in human than in horse hemoglobin, although the difference may be smaller than reported earlier. As in carbonylhemoglobin, one or two other more basic groups per heme are also masked in the native low-spin cyanoferriheme protein. It is shown that reversible changes in absorbance (Soret band) incident to unfolding are paralleled by changes in optical rotation (both Soret region and far ultraviolet). © 1969.