MECHANISMS OF LIGAND BINDING TO HEMOGLOBIN
REISBERG, PAUL IRWIN
Doctor of Philosophy
The reactions of thirteen isonitriles with hemoglobin, and its isolated subunits were characterized in order to examine the effects of ligand size and stereochemistry on hemoglobin function. All of the isonitriles bind to hemoglobin in a cooperative manner. The exact amount of cooperativity decreases with ligand length but increases with increasing substitution on the alkyl side chain. The affinities of hemoglobin for these compounds exhibit a more complicated dependence on stereochemistry, which has been interpreted in terms of competing hydrophobic and steric interactions. Subtraction of hydrophobic effcts from the observed free energy changes of ligand binding allowed the construction of a rough map of steric hindrances at the heme site. The association and dissociation rates of the high affinity forms of hemoglobin (dimers and isolated chains) have also been examined in terms of bound free energy potentials as above and in terms of kinetic barrier potentials. For the n-series, the observed association rates exhibited a V-shaped dependence on ligand length with methyl and n-hexyl isocyanides binding at the fastest rates and n-propyl the slowest. The large association rates for the longer isonitriles argue against the concept of a small solvent channel limiting the entrance of ligands into the heme site. Rather, it appears that the ligand molecules rapidly dissolve within the hydrophobic interior of the protein and then diffuse to the heme group. Access to the iron atom appears to be attenuated by its coordination geometry and by local steric interactions. Kinetic studies using native hemoglobin showed that the isonitrile ligands can be divided into two mechanistic classes. (1) The larger ligands ((GREATERTHEQ) butyl) exhibit biphasic time courses at all ligand concentrations; this is the result of the (beta) subunits within the low affinity form of hemoglobin reacting with ligand 5-75 times faster than the (alpha) subunits. (2) The smaller isocyanides, exhibit biphasic time courses only at low ligand concentrations where the large differences between the dissociation rate constants for the high and low affinity conformations are expressed; at higher concentrations monophasic or accelerating behavior is observed. A complete description of the binding of each of the isonitriles to human deoxyhemoglobin was obtained by fitting simultaneously an equilibrium curve and a set of kinetic time courses to an expanded 2-state allosteric model. The rate and affinity constants of the R (high affinity) and T (low affinity) states were used to calculate potentials for the ligands at the kinetic barrier and bound to the heme iron atom within the individual (alpha) and (beta) subunits of each protein conformation. The R state free energies were virtually identical to those determined for the isolated (alpha) and (beta) chains, whereas the T state values were 1 - 4 Kcal/mole higher. For (beta) subunits, the barrier and bound potential differences between the R and T states were independent of ligand length and stereochemistry. Thus steric interactions at the binding site exert little or no effect on the expression of cooperativity by this subunit. The differences between the R and T potentials for the (alpha) subunits are larger so that a greater part of the overall observed cooperativity is due to these subunits. The magnitude of these differences decreases with increasing ligand length, which suggests that steric effects are important for the expression of cooperativity by (alpha) subunits.