The pK of the amino terminal groups of carbonmonoxy- and deoxyhemoglobin measured by dinitrophenylation in phosphate buffers.

The rate of reaction of the terminal valines of the alpha- and beta-chains of hemoglobin with 1-fluoro-2,4-dinitrobenzene was followed spectrophotometrically at 353 nm. The variation with pH of the rate of dinitrophenylation of these groups was measured for both carbonmonoxy- and deoxyhemoglobin. In carbonmonoxyhemoglobin the results indicated a pK near 6.7 and 7.7 for the amino terminal groups of the two kinds of subunits, and were attributed to the alpha- and beta-chains respectively. Removal of ligands produced an increase of 0.1 in both pK values and a decrease of 40% of the pH-independent kinetic constant for dinitrophenylation of the beta-subunits. These modifications are due to the conformational changes associated with ligand binding in the system. In phosphate buffers the contribution to the Bohr effect of the amino terminal residues of either chains is negligible.