Kinetic Studies on the Five Principal Components of Normal Adult Human Hemoglobin

The five principal components of human hemoglobin (A_1a, A_1b, A_1c, A₀, and A₂) have been isolated by column chromatography and by preparative isoelectric focusing in gels. The isoelectric points and a number of kinetic parameters have been determined for each hemoglobin. The greatest kinetic differences are found in the binding of CO to the deoxy conformation. At pH 7, A₀and A₂are nearly identical in their overall reaction with CO, whereas the initial lag phase characteristic of crude hemolysate and A₀is greatly reduced in A_1aand A_1cand is essentially absent in A_1b- The general effect of p-mercuribenzoate binding on CO association is to magnify kinetic differences among the hemoglobins, diminish the initial lag phase, and increase the overall rate of CO binding. Hemoglobin A_1ais anomalous in that the overall CO binding rate actually decreases after reaction with the mercurial. In terms of an Adair model with four association constants the rate constant for the binding of the first molecule of CO (1_l′) showed the greatest variation among the five hemoglobins, with A₀having the smallest constant, and A_1bthe largest. For the native hemoglobins, 1_l′ for A_1bwas more than twice that for A₀; for the mercurated hemoglobins, the difference was greater than threefold. Raising the pH from 7 to 8 increases 1_l′ for all hemoglobins, but A_1ais anomalous in having a slower overall rate for CO binding at the higher pH. At pH 9, the time course of CO binding is biphasic for all hemoglobins, with A₀, the fastest, and A_1a, the slowest, differing by nearly threefold in rate. The equilibrium constant for the tetramer-dimer equilibrium was determined by flash photolysis. The largest dissociation constant occurs for A_1aand is 4.4 times that for A₀, and 5.6 times that for A_1c, the least dissociated of the hemoglobins. The overall oxygen dissociation reaction is biphasic for A_1aand A_1b, with the two phases differing by a factor of 5; the dissociation reactions for the other three hemoglobins appear essentially monophasic. The kinetics of dissociation of the first oxygen molecule from oxyhemoglobin are very similar for all five hemoglobins, as are the association kinetics for CN^-and N₃ ^-binding to the five methemoglobins.