Reaction of 1-Fluoro-2,4-dinitrobenzene with the Free α Chains of Human Hemoglobin

The reaction of 1-fluoro-2,4-dinitrobenzene with isolated α chains in their mercaptide form with p-mercuribenzoate (PMB) was studied. From kinetic measurements with the change in absorbance at 353 nm upon substitution of the terminal valine, the pK of this group was calculated to be 7.30. The pH-independent reaction constant was estimated to be 0.282 m-1 sec-1. Comparing the proton-binding behavior of untreated α chains with that of the chains substituted in the terminal valine (αpmbDNP) the pK of this group was calculated to be 7.4. Histidines did not appear to be dinitrophenylated. However, from the rate of liberation of protons upon the reaction of the chains with 1-fluoro-2,4-dinitrobenzene we concluded that they do interact with the reagent without forming a stable derivative. A pK of 6.85 and a pH-independent reaction constant of 0.023 m-1 sec-1 per individual group was calculated for these residues. From oxygen equilibrium studies on a mixture in equal amount of αpmbDNP and β chains in their mercaptide form with p-mercuribenzoate we calculated a value of 1.35 for the Hill parameter n. The s20,w of this mixture was 4.95 while it is near 2.5 when the α chains are not dinitrophenylated (Antonini et al., J. Mol. Biol., 17, 29 (1966)), indicating that the dinitrophenylation strongly favors the formation of tetramers. This high sedimentation value is consistent with a partial specific volume v = 0.731, as compared to v = 0.749 commonly used for hemoglobin (Svedberg and Pedersen, The ultracentrifuge, Oxford University Press, New York, 1940).