Equilibrium binding of alkyl isocyanides to human hemoglobin.

The reactions of human hemoglobin with a series of 13 alkyl isocyanides have been examined in equilibrium titration experiments at pH 7, 20 degrees C. The ligands include: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, (+)- and (-)-sec-butyl, tert-butyl, n-pentyl, n-hexyl, cyclohexyl, and benzyl isocyanides. All of these compounds exhibit sigmoidal binding curves; however, the amount of cooperativity expressed decreases with ligand length and increases as the alkyl side chains become more highly substituted. The overall affinity of hemoglobin for these compounds also exhibits a complex dependence on ligand size and stereochemistry. These results have been interpreted in terms of competing favorable hydrophobic interactions and unfavorable protein steric effects. The overall chemical potentials of the bound ligands were calculated from the sum of the observed binding free energy change and the relative chemical potentials of the isonitriles in aqueous solution. The resulting values allowed the construction of a rough, three-dimensional free energy map of steric hindrance at the sixth coordination position of the heme iron atom. This scheme suggests a cylindrical cavity of weak protein interactions into which ethyl isocyanide can easily fit. This cavity or mobile region of protein structure is surrounding by a more rigid region which results in large unfavorable steric interactions. Finally, this ring of more rigid structure is followed by an outer area where considerably smaller steric hindrance effects are observed.