FURTHER STUDIES ON HEMOGLOBINOXYGEN EQUILIBRIUM

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Abstract
The dependence of the heme-heme interaction of human adult hemoglobin on the species and strength of the environmental salts was studied. In a series of several neutral salt solution, the magnitude of the interaction was dependent not only upon the ionic strength but also upon the species of the environmental salt. In the latter of the 2 factors, the charge of the constituent cation appears to have a primary importance. All these results confirm our previous observations. Similar effects were also verified in potassium phosphate buffer (pH 6.9). The magnitude of the effect was roughly comparable to that of the divalent cation neutral salts. An attempt was made to graphically estimate the intrinsic constants of the 1st and 4th O2 association (k1 and k4 by human adult hemoglobin. The k4 value was 170 to 250 times as great as the k1 in NaCl ([mu]=1.0), whereas the enhancement was only 14 to 25 fold in no salt medium. Oxygenation of hemoglobin was accompanied with a hyperchromicity and a slight blue shift in the medium ultraviolet range. These changes were completely reversible. The derived ultraviolet difference spectrum (oxy vs. decay subtraction) showed 3 different peaks around 238,275, and 290 m[mu]. A linear correlation was shown between the degree of oxygenation and the magnitude of the ultraviolet spectral change. Deoxygenation of bovine oxy-hemoglobin induced a remarkable reduction in the alkali resistance. Discussions were made of a probable correlation of the alterations in the ultraviolet absorption and the alkali denaturation with a configurational change of hemoglobin molecule upon the oxygenation.